Human Reproduction vol.14 no.2 pp.505–514, 1999

Immunohistochemical localization of receptors forprogesterone and oestradiol-17β in the implantation siteof the rhesus monkey

Debabrata Ghosh, Surajit Dhara, Arvind Kumarand Jayasree Sengupta1

Department of Physiology, All India Institute of Medical Sciences,New Delhi 110029, India1To whom correspondence should be addressed

The aim of the present study was to examine the cellularbasis of the involvement of oestradiol and progesterone inblastocyst implantation in the primate. To this end, thecellular distribution of receptors for oestradiol (ER) andprogesterone (PR) in fetal trophoblast cells and in endo-metrial compartments of timed lacunar (pre-villous) andvillous stages of placentation in primary implantation sitescollected on days 13–22 of gestation were investigated inrhesus monkeys. Both in pre-villous stage tissue and invillous stage tissue, cytotrophoblast cells and syncytiotro-phoblast cells and other trophoblast derived cells were PRpositive, while they were generally ER negative. Maternalendometrial cells were ER negative, while epithelial cells,stromal cells and vascular endothelial cells in maternalendometrium showed heterogeneous staining patterns forPR depending on their relative location; these patterns,however, correlated well with glandular hyperplasia anddifferentiation, stromal–decidual transformation and vas-cular response seen during blastocyst implantation.Key words:cytotrophoblast /endometrium/oestrogen receptor/progesterone receptor/syncytiotrophoblast

Introduction

Macroscopic and microscopic characteristics of implantationand placentation stages in humans have been described byHertig and Rock (1944) and in the macaque by Wislockiand Streeter (1938). The ultrastructural features of initialpenetration by trophoblast cells of maternal tissues (Reniuset al., 1973; Enderset al., 1983; Enders and King, 1991),formation and differentiation of extraembryonic mesoderm(Luckett, 1978; Enders and King, 1988) and the nature ofendometrial responses to trophoblast penetration (Enderset al.,1985) in the macaque have also been described. However, theendocrine equivalents of trophoblast invasion and placentationin a stage-specific manner are not known for the rhesusmonkey in particular, and for primates in general. Elevatedconcentrations of oestrogen, progesterone and the presence ofchorionic gonadotrophin and relaxin in the systemic circulationare the associated endocrine features of conception cycles inwomen and in macaques (Sekiet al., 1985; Stewartet al.,1993; Roberts and Anthony, 1994; Ghoshet al., 1997). There

© European Society of Human Reproduction and Embryology 505

is evidence to suggest that progesterone is essential forsupporting implantation associated events in the uterus, whileoestradiol from ovarian sources may not be required forimplantation in non-human primates and women (Ghoshet al.,1994; Zegers-Hoshchild and Altieri, 1995). However, oestra-diol from embryonic and endometrial sources (Tsenget al.,1986; Edgaret al., 1993) may still play a permissive rolein embryo implantation (Ghosh and Sengupta, 1995). Tounderstand the cellular basis of hormone action, it is useful toinvestigate the cellular distribution of receptors for steroidhormones. The use of monoclonal antibodies against receptorsfor oestradiol (ER) and progesterone (PR) permits cell-typespecific localization of steroid receptors (Presset al., 1984;Press and Greene, 1988). Through such studies, it has beenreported that ER and PR show cyclical variations in endometrialglandular and stromal cells during the menstrual cycle (McClel-lan et al., 1984; Presset al., 1984; Lesseyet al., 1988; Pressand Greene, 1988; Amsoet al., 1994). In the present studythe immunolocalization of ER and PR in fetal trophoblast cellsand in cells of the maternal endometrial compartment duringlacunar and villous stages of placentation in the rhesus monkeywas investigated.

Materials and methods

Animals

The details of animal management are given elsewhere (Ghoshet al.,1993, 1996, 1997). Briefly, proven fertile male and female rhesusmonkeys were housed singly under semi-natural conditions in thePrimate Research Facility of the All-India Institute of MedicalSciences, and were fed with regular monkey pellet diet, semi-formulated Indian bread and fresh seasonal fruits, and wateradlibitum. Females were allowed to cohabit during days 8–16 of theirmenstrual cycles with males, and during this time twice dailyperipheral blood samples were collected from female animals for theassessment of oestradiol-17β, progesterone and chorionic gonadotro-phin (CG) concentrations in peripheral circulation using radioimmuno-assay techniques in order to detect the day of ovulation (day 0) asdescribed previously (Ghoshet al., 1993, 1996, 1997). Vaginal smearswere checked daily for the presence of spermatozoa. The experimentaldesign of the present study was approved by the Ethics Committeeon the Use of Non-Human Primates in Biomedical Research of theAll-India Institute of Medical Sciences.

Tissue collection and processing

Prediction of pregnancy was made on the basis of elevated profilesof oestradiol-17βand progesterone and detectable CG in peripheralcirculation (Ghoshet al., 1997). On estimated days 13–22 (n 5 14)post-fertilization, animals were laparotomized under ketamine (12 mg/kg body weight; Parke Davis & Co., Mumbai, India) anaesthesia andafter checking for the presence of functional corpus luteum, in-situ

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Table I. Description of implantation sites recovered from rhesus monkeys

Animal Estimated day Important microscopic featuresnumber of gestation

2459 13 Layer of extraembryonic trophoblast cells;2367 13 cytotrophoblast cells lining extraembryonic cavity;

Syncytiotrophoblast cells lining developinglacunae; maternal blood cells within lacunae;trophoblast plate below lacunae; embryonic disc;very few extraembryonic mesodermal cells; dilatedvenules at maternal–fetal border withhypertrophied vascular endothelial cells, and fewtrophoblast cells located within and adjacent toblood vessels; plaque acinar cells adjacent toimplantation zone and at the border of oedematouszone. See Figure 1A, a.

2444 14 Expanded implantation site with wide lacunae;2305 14 embryonic disc with well defined columnar2417 15 ectodermal cells and endodermal cells forming2150 15 periphery of disc; squamous endodermal cells2141 16 continuous with lining of vitelline sac; primary2110 16 villi, secondary villi by days 15–17; non-polarized2352 16 cytotrophoblast cells forming columns at bases of2231 17 villi, lined by syncytiotrophoblast cells;

interstitial trophoblast cells at fetal–maternalborder, groups of trophoblast cells within dilatedvenules, endovascular trophoblast cells withinspiral arterioles in principal zone of endometrium;decidual cells interspersed with fibroblastic stromalcells in fetal–maternal border and in the upper partof principal zone of endometriumbelow implantation site; plaque acini at lowerborder of implantation site, plaque cellsdegenerating. See Figures 1B, b, C.

2308 22 Considerable placental expansion; anchoring villi2237 22 and floating villi below chorion; distinct2318 22 cytotrophoblast shell with areas of necrotic cells;2248 22 non-polarized cytotrophoblast cell columns lined

by syncytiotrophoblast cells extending into shell;distal ends of anchoring villi contributing groupsof syncytiotrophoblast cells to the trophoblastshell; distinct layer of decidual cells immediatelybelow placental villi and on both sides; extensivevascular invasion by trophoblast cells. SeeFigure 1D.

perfusion was performed using sterile phosphate buffered saline(pH 7.4) followed by freshly prepared 4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4. In-situ perfusion fixation was performedfor 45–60 min, then the uterus was quickly excised and placed infresh fixative on ice for transportation to the laboratory. In thelaboratory, the uterus was carefully opened to expose its luminalsurface, the primary implant site was located, and carefully excisedand kept for fixation for 24 h at 4°C and then processed in a routinemanner for paraffin embedding (Ghoshet al., 1993, 1996, 1998).

Analysis of implantation sites and endometrial compartments

Based on light microscopic examination of haematoxylin stainedparaffin sections, the implantation stages for each sample weredocumented based on earlier descriptions (Hendrickx and Houston,1976; Houston, 1976; Enders, 1993). Implantation sites and associatedfetal and maternal compartments were analysed using the zonationdescription given by Enders and King (1991). According to thisdescription, these are: superficial zone, principal zone, transitionalzone and basal zone. The superficial zone includes luminal epitheliumand subepithelial compartment containing first part of glands andstroma, and implanted embryo. The principal zone forms the majorpart of endometrium, and is occupied by the straight part of glands,and in the narrow transitional zone by coiled glands. Basal zone

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contains glands characterized by tall columnar epithelia. Immunohisto-chemical localization for cytokeratin, vimentin, and von Willebrandfactor (vWf) was performed on parallel sections to distinguishtrophoblast cells, mesenchymal–stromal–decidual cells and endothel-ial cells respectively at implantation sites.

Immunohistochemistry

Paraffin blocks were serially sectioned at 5µm using a Supercutmicrotome (Leica, Germany) and sections were collected on poly-l-lysine precoated glass slides. Immunohistochemical localization forER and PR was performed using mouse monoclonal antisera againstER and PR (Immunotech, Cedex, France) respectively. ER-specificmonoclonal antibody, ER/D5 was produced by using spleen cellsfrom BALB/c mice immunized with recombinant ER protein, andPR-specific monoclonal antibody, PR10A9 was produced against asynthetic peptide that contains the carboxy-terminal 12 amino acidsof PR. Immunodetection of ER and PR were performed using amicrowave method of antigen retrieval (three heating cycles of 5 mineach in 0.1 M citrate buffer, pH 6.0) and trypsinization (0.05%, w/vfor 10 min) as described by Szekereset al. (1994a,b) and Nayaket al. (1998). For both ER and PR, two methods (i.e. microwaveretrieval followed by trypsinization, and trypsinization followed bymicrowave retrieval) were performed on serial sections. Additionally,we examined whether protein unmasking by the above retrievaltreatments could result in enhanced tissue peroxidase activity andbiotin activity, since a biotin–avidin–peroxidase method was employedfor final visualization. In order to examine the peroxidase activity,following retrieval treatment, the sections were post-treated withhydrogen peroxide (0.3%, v/v) in phosphate buffered saline (pH 7.4)and used for PR and ER immunohistochemistry (Finley and Perutz,1982). To examine the avidin-binding activity, sections were treatedwith avidin (0.01%, w/v) followed by biotin (0.001%, w/v) asdescribed by Fisheret al. (1997) and subjected to immunohistochem-istry for ER and PR. Sections were incubated in primary antibodyovernight at 4°C. This was followed by incubation with biotinylatedsecondary antibody and final visualization was achieved by using theABC peroxidase kit (Vector Laboratories, Burlingame, CA, USA)and freshly prepared 3,39-diaminobenzidine tetrahydrochloride andhydrogen peroxide according to the protocol provided by the manufac-turer. Since immunopositive stainings for ER and PR were detectedprimarily in nuclear compartment and occasionally in cytoplasm, nocounter staining was done in these sections.

It was observed that staining was relatively discrete and moreintense with the method of microwave retrieval followed by trypsiniz-ation for both ER and PR compared with the staining performedusing a method in which microwave retrieval in citrate buffer wasdone after trypsinization. Incubation with hydrogen peroxide followingmicrowave retrieval and trypsinization substantially reduced non-specific staining, mainly in cytoplasmic area, for both ER and PR.Additional treatment with avidin to block tissue avidin-binding activityexposed by retrieval treatment failed to reduce any further non-specificity in the staining pattern. Thus, sections subjected to micro-wave retrieval treatment in citrate buffer followed by trypsinization,post-treated for quenching of peroxidase and avidin-binding activities,and finally immunohistochemically stained with respective mono-clonal antibodies for ER and PR were analysed for their immunoposi-tive distribution in different fetal and maternal compartments duringpre-villous and villous stages of placentation.

As mentioned above, parallel sections of implantation sites fromeach sample were processed for immunocytochemical localization ofcytokeratin, vimentin and vWf using specific monoclonal antisera(Dako-CK MNF116, Dako-vimentin V9, and Dako-vWf F8/86respectively) obtained from Dako A/S (Glostrup, Denmark) and final

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visualization was obtained using the ABC peroxidase kit and freshlyprepared diaminobenzidine hydrochloride with hydrogen peroxide asdescribed above. These sections were counter-stained with haema-toxylin.

Dilutions of stock primary antibodies for incubation were precalib-rated based on 3–5 points titration and the information provided bythe manufacturers. Specificity of antibody liganding and visualizationwere assessed by (i) omitting primary antibodies, (ii) replacingprimary antibodies with unrelated immunoglobulins from same speciesand other species, (iii) omitting secondary antibodies, and (iv)replacing labelled secondary antibody with unrelated labelledimmunoglobulins from same species and other species. For a givenprobe, all sections were subjected to immunohistochemistry simultan-eously. Late proliferative and early luteal phase endometrial tissuesections were used as positive controls. Labelled and unlabelledimmunoglobulins, non-immune sera, and visualization kits (horserad-ish peroxidase) were purchased from Vector Laboratories. All otherchemicals were purchased from Sigma Chemical Co. (St Louis,MO, USA).

For assessment of immunostaining in cells of embryonic andendometrial compartments, semiquantitative subjective scoring wasdone based on a 5-scale system: 0, nil (0%); 1, very weak (, 5%);2, weak (5–25%); 3, moderate (25–75%); 4, strong (. 75%), asdescribed by Presset al. (1988). It was assumed that these measure-ments reflect the concentrations of the experimental protein in differentfetal and endometrial compartments.

Results

Table I gives the detailed description of samples investigatedin the present study. Figure 1 shows representative lacunar

Figure 1. Representative micrographs of haematoxylin stained,paraffin sections of implantation sites obtained from: (A) animal2459 on estimated day 13 of gestation. Bilaminar embryonic disc(ED), cytotrophoblast and syncytiotrophoblast cells forming anexpanded trophoblast plate (TP), small clefts and trophoblasticlacunae (L) are seen in this region. The uterine epithelium iscompletely missing over the area of contact between embryo andendometrium. A dilated blood vessel (BV) continuous withtrophoblast lacunae immediately below the implantation site,maternal red blood cells can be seen within lacunae. At highermagnification (a), ED is characterized by loosely arranged cells ofepiblast (ep) and endoderm (end). (B) Animal 2444 on estimatedday 14 of gestation. Well defined bilaminar embryonic disc (ED),intervillous spaces (IVS) and plaque acini (PL) are seen. In highermagnification (b), ED is characterized by epiblast plate (ep)bordering the amniotic cavity (am) and endodermal layer (end)bordering the primitive vitelline cavity (vit). (C) Animal 2417 onestimated day 16 of gestation. The cells of epiblast havedifferentiated into pseudostratified columnar ectodermal cells (ect),and thin endodermal cell layer (end). A larger amniotic cavity (am)and a smaller vitelline cavity (vit) are present. The placenta iselevated from uterine surface and protrudes into uterine cavity. Athin membraneous chorion (CH) with an inner mesothelial layerextends from edge of placenta into uterine cavity. Cytotrophoblastcell columns (CC) from the tips of open villi to the base ofplacenta are distinct. Numerous dilated BV are continuous withintervillous spaces (IVS) which are filled with maternal blood.(D) Animal 2318 on estimated day 22 of gestation. Distinctlybranched chorionic villi are seen. Thick cytotrophoblastic shell(SH), darkly stained junctional zone (JCT) containing trophoblastcells, necrotic cells and decidual cells, an area of decidua basalis(DB) are seen. Bars5 300 µm (A, B, C, D) and 1000µm (a, b).

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(pre-villous) and villous stages as obtained in the presentstudy. Table II shows the immunohistochemical distributionof cytokeratin and vimentin in pre-villous and villous stageimplantation sites on days 13–22 of gestation. Immunolocaliz-ation revealed cytokeratin-positive staining in all populationsof trophoblast cells (Figure 2a–d). Intense immunopositivestaining for cytokeratin was observed in syncytiotrophoblast(STB) cells lining lacunae and villi, and moderate staining forcytotrophoblast (CTB) cells present as non-polarized columnsof cells. Venules and spiral arterioles in principal zone wereoften infiltrated with cytokeratin-positive trophoblast cells;interstitial trophoblast cells were also cytokeratin-positive

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(Figure 2c). Extraembryonic mesenchymal cells were vimentin-positive (Figure 2e, f), villous mesenchymal cells were alsodiscretely cytokeratin-positive (Figure 2d). Vimentin-positiveendometrial stromal cells surrounding plaque acini andadjoining dilated blood vessels (Figure 2g, h) were seen.Within the implantation site, vimentin-positive stromal cellswere often detected as discrete groups of cells at the lowerend of the trophoblast plate (Figure 2g, h). Vimentin-positivestromal cells were also observed in the superficial zone onboth sides of the implant. Decidual cells adjacent to the CTBshell were positive for vimentin. At this stage of placentaldevelopment, blood vessels – mainly venules – were dilated,

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and vWf-positive vascular endothelial cells (VEC) were mark-edly hypertrophied at implantation sites (Figure 2i).

Figures 3–5 show immunolocalization of PR and ER indifferent cell types of embryonic and endometrial compart-ments during pre-villous and villous placentation. Tables IIIand IV provide the scores of immunopositive PR in cells offetal and endometrial compartments at implantation sites andendometrial cells adjacent to implantation sites, respectivelyin 10 tissue samples collected during days 13–17 of gestation.On the fetal side, CTB cells lining embryonic cavity, CTBand STB cells lining lacunae and villi, and trophoblast cellsin trophoblast plate were PR positive (Table II; Figures 3a,4a). Polarized CTB cells associated with lacunae and villiwere generally more intensely PR positive compared with STBcells and non-polarized CTB cells of the column. On theendometrial side, only stromal cells in all zones, and glandularepithelial cells in transitional and basal zones, and few venularVECs at implant sites, and vascular smooth muscle (VSM)of spiral arterioles in superficial and principal zones, werecharacteristically PR positive (Tables III and IV; Figures 4b,5). Generally, decidual cells at implantation sites showed less

Table II. Immunohistochemical distribution of cytokeratin and vimentin inpre-villous and villous stage implantation sites on days 13–22 of gestation*

Cell type Scorea

Cytokeratin Vimentin

Fetal:STB and CTB lining lacunae/villi 4 (3–4) 0 (0)Extravillous trophoblast cells 4 (3–4) 0 (0)Amniochorion 4 (2–4) 1 (0–2)Villous mesenchyme 2 (0–2) 4 (3–4)

Maternal:Gland epithelium 4 (3–4) 0 (0)Plaque epithelium 4 (3–4) 0 (0)Stromal fibroblasts 0 (0) 4 (3–4)Decidual cells 0 (0) 2 (1–3)

*n 5 14. aMedian values with ranges based on 5-scale subjective scoringmethod as described in text. STB5 syncytiotrophoblast, CTB5cytotrophoblast.

Figure 2. Micrographs of immunohistochemically stained sections for cytokeratin, vimentin and vWf. (a) Immunopositive cytokeratinstaining of trophoblast cells of implantation site. Cytotrophoblast cells (CTB) lining embryonic cavity and syncytiotrophoblast cells (STB)lining lacunar space (L), trophoblast cells of plate region (TC) are positive; maternal stromal cells are negative. Bar5 50 µm. (b)Trophoblast-lined villi, trophoblast plate and fetal–maternal border of implantation site immunostained for cytokeratin. STB,Syncytiotrophoblast cells lining secondary villi; CC, columns of cytotrophoblast cells. Groups of trophoblast cells present as a migratorycolumn of cells at the fetal–maternal border of implantation site (X), interstitial trophoblast cells (arrows) and plaque cells (arrow heads)are cytokeratin-positive. Bar5 80 µm. (c) Implantation site recovered showing immunostaining for cytokeratin at the fetal–maternal border.Maternal stromal cells are negative. Migratory columns of trophoblast cells (X) are positive. Cytokeratin-positive interstitial trophoblast cellspresent within maternal stroma (arrows), and within maternal blood vessel (arrow head). Bar 5 50 µm. (d) Secondary villi showingimmunostaining for cytokeratin. Syncytiotrophoblast cells (STB) and cytotrophoblast cells (CTB) lining villi show positive staining. Notethe delicately positive stain in extraembryonic mesodermal cells (arrows). Bar 5 50 µm. (e) Implantation site immunostained for vimentin.Trophoblast cells are negative, extraembryonic mesodermal cells are positive, epiblast (EP) and endodermal cells (END) are negative,loosely arranged outer mesothelial layer (arrows) is positive. Bar5 50 µm. (f) Immunohistochemical staining for vimentin of implantationsite. Extraembryonic mesodermal cells (arrows) adjoining primary villi and within secondary villi are postively stained. Bar5 50 µm. (g)Vimentin immunostaining in maternal stromal cells of implantation site. Implantation zone lying below lacunae (L) shows that trophoblastcells are negative, while stromal cells stain intensely for vimentin. Bar5 50 µm. (h) Vimentin immunostaining of cells adjoiningimplantation site. Plaque cells (PL), luminal epithelia (LE) are negative, stromal cells located within and surrounding plaque acini and bloodvessel are strongly positive. Bar5 80 µm. (i) von Willebrand factor immunostaining of endothelial cells (arrows) of dilated blood vessels atfetal–maternal border. Plaque cells (PL) and stromal cells are negative. Note blood vessels present in plaque interacinar spaces (arrow head)and extracellular matrix surrounding the blood vessels are immunopositive. Bar5 80 µm.

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intense PR positivity compared with stromal cells in associatedareas. Some intravascular trophoblast cells showed cytoplasmicstaining (Figure 4b). Since there was little immunodetectableER cell types in the samples of gestation days 13–17, data arenot shown (see Figure 3c). Table V shows the scores ofimmunopositive PR and ER in cells of fetal and endometrialcompartments at implantation sites from four samples collectedon day 22 of gestation. In these samples, the overall dis-cernibility of positive signals was generally higher comparedwith earlier stages, however, there was no change in thebasic pattern of immunostaining for ER and PR in fetaland endometrial sides; CTB and STB cells surrounding villi(Fig. 4c), interstitial trophoblast cells and trophoblast cells inCTB shell (Figure 3b), intravascular trophoblast cells(Figure 4d), and stromal cells in endometrium showed PRpositivity. Except for occasional trophoblast cells (Figure 3d),cells in both compartments were ER negative.

Discussion

The light microscopic examination of primary implantationsites during early stages (days 13–22) of pregnancy of therhesus monkey revealed the following observations. In samplesof day 13 of gestation, lacunae of different dimensions wereevident in trophoblast plates. Distinctive endometrial responsesto invading trophoblasts, which included subepithelial oedema,and epithelial plaque cells adjacent to implantation sites wereevident. On subsequent days, the expansion of lacunae wasfollowed by formation of primary villi (by days 14–15 ofgestation) and then secondary villi (days 15–17) and tertiaryvilli (day 22), and was associated with substantial expansionof implantation sites. At this time, oedema was reduced,regressive changes in plaque cells became distinctive, andstromal cell recruitment to decidual transformation wasenhanced. Throughout this period, venular dilatation, bloodvessel penetration by trophoblast cells, and vWf-positiveendothelial cell hypertrophy were remarkable features atimplantation sites. These observations are very similar to thosereported earlier in the same species (Wisloki and Streeter,

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Figure 3. Immunohistochemical staining for PR and ER ofimplantation sites. (a) Maternal stromal cells are positively stainedfor PR, but stromal cells immediately bordering trophoblast cells(bracket) showing weak staining, and stromal cells adjoiningimplantation site (arrow) are intensely stained. Cytotrophoblastcells (CTB) bordering embryonic cavity are positive,syncytiotrophoblast cells (STB) are weakly stained. Endothelialcells of dilated blood vessel show weak cytoplasmic staining(arrow head). Glandular epithelial cells (GE) are negative. Bar580 µm. (b) Basal plate of placenta at 22 days of gestationimmunostained for PR. Cytotrophoblast shell (SH) and junctionalzone (JCT) comprising trophoblast cells and decidual cells areimmunopositive for PR. Endothelial cells of dilated blood vessels(BV) show both nuclear and cytoplasmic immunostaining. Bar580 µm. (c) ER immunostaining in implantation site recovered onday 15 of gestation showing no positive immunostaining introphoblast cells and endometrial cells of the uterus. Bar5 80 µm.(d) ER immunostaining in interstitial trophoblast cells locatedbelow anchoring villi and within the junctional zone of basal plateof placenta at 22 day of gestation. Immunostaining is seen incytoplasmic and nuclear compartments. Bar5 50 µm.

1938; Heuser and Streeter, 1941; Enderset al., 1983, 1985;Enders and Schlafke, 1986; Enders, 1993). Immunolocalizationof cytokeratin and vimentin in these samples revealed that alltrophoblast cells were cytokeratin-positive including STB andCTB lining lacunae and villi, interstitial trophoblast cells andendovascular cytotrophoblast cells (EVCT), while mesenchy-mal cells of the amniochorion and chorionic villi were positivefor both vimentin and cytokeratin. Similar observation hasbeen reported earlier in human samples collected in week 36of gestation (Khonget al., 1986) and in monkey samplescollected on days 22–160 of gestation (Blankenshipet al.,

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Figure 4. PR immunostaining in trophoblast cells lining villi, andcytotrophoblast cell column (CC), and extraembryonic mesodermalcells (arrows) of implantation site recovered on day 16 ofgestation (a), in endothelial cells (arrows ) of dilated blood vessel(BV) at fetal–maternal border, and in intravascular trophoblast cells(arrow heads) (b), in placental villi at 22 days of gestation withcytotrophoblast cells (CTB) showing stronger immunostainingcompared with syncytiotrophoblast cells (STB), and non-polarizedcytotrophoblast cells of cell column (CC) showing weak andinfrequent staining (c), in trophoblast cells (*) within maternalblood vessel in endometrium at 22 days of gestation (d). Note thepositive immunostaining in endothelial cells (arrow) (d). Bars545 µm (a), 30 µm (b, c) and 20µm (d).

1993). It is to be noted that the purpose of immunostainingfor cytokeratin, vimentin and vWf was to identify trophoblastcell, mesenchymal cell and endothelial cell populations inmaternal endometrial stromal compartment, especially at lociwhere these cell types co-exist in close juxtaposition and thatwe made no attempt to examine the specificity of cytokeratin-type expressed by different subsets of trophoblasts accordingto location and stage of differentiation. Indeed, there is evidencethat differentiation of human trophoblast cell populationsinvolve alterations in the patterns of cytokeratin expression(Muhlhauseret al., 1995; Vicovac and Aplin, 1996).

We now demonstrate for the first time the pattern ofdistribution of PR and ER in the embryonic compartment,placenta and maternal endometrium during early pregnancy(days 13–22 of gestation) of the rhesus monkey. It is generallybelieved that knowledge of cellular distribution of receptors for

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Figure 5. PR immunostaining of implantation site associatedendometrium. Stromal cells in oedematous area adjacent to theimplant in superficial zone are positive (a). In the principal zone,stromal cells are positively stained, while glandular epithelial cells(GE) are negative (b). In the transitional zone, glandular epithelialcells (GE) express heterogeneity in staining for PR; stromal cellsstaining for PR were less intensely stained than that in the principalzone (c). Glandular epithelial cells (GE) and stromal cells in basaliszone are positively stained(d). Bar 5 45 µm.

Table III. Immunohistochemical localization of progesterone receptors inpre-villous and villous stage implantation sites on days 13–17 of gestation*

Cell type Scorea

Fetal:Extraembryonic trophoblast cells 0 (0–1)Embryonic disc 0 (0–1)Extraembryonic/villous mesenchyme 1 (0–2)CTB cells lining embryonic cavity 2 (1–3)STB cells lining lacunae 2 (1–2)CTB cells lining primary and secondary villi 3 (2–4)STB cells lining primary and secondary villi 2 (1–4)Intermediate trophoblast cells 2 (1–4)Trophoblast cells within dilated venules in implantation site 1 (1–3)EVCT within arteriole in principal zone 1 (1–3)Maternal:Plaque acinar cells 0 (0–1)Decidual cells 2 (1–4)Fibroblast cells at maternal–fetal border 3 (2–4)Modified endothelium of dilated venules 2 (0–4)

*n 5 10. aMedian values with ranges based on 5-scale subjective scoringmethod as described in text. CTB5 cytotrophoblast, STB5syncytiotrophoblast, EVCT5 endovascular cytotrophoblast cells.

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Table IV. Immunohistochemical localization of progesterone receptors inendometrial cells of pre-villous and villous stage implantation sites ondays 13–17 of gestation*

Cell type Scorea

Luminal epithelium 0 (0–1)Glandular epithelium

Superficial, principal 0 (0–2)Transitional 1 (0–3)Basal 3 (1–4)

Decidual cells 2 (1–4)Stromal cells

Superficial, principal, 3 (2–4)Transitional, basal

Blood vesselsSuperficial, principal 1 (0–2)Transitional, basal 0 (0–1)

*n 5 10. aMedian values with ranges based on 5-scale subjective scoringmethod as described in text.

Table V. Immunohistochemical staining for progesterone receptors (PR) andoestrogen receptors (ER) in fetal and maternal cells at implantation sites onday 22 of gestation*

Cell type Scorea

PR ER

Fetal:Chorion 1 (1–2) 0 (0–1)Extraembryonic mesenchymes 1 (1–3) 0 (0–1)CTB cells in floating villi 3 (2–4) 0 (0–1)STB cells in floating villi 3 (1–4) 0 (0–1)Non-polarized CTB cells forming columns 1 (0–2) 0 (0–2)STB cells lining CTB cell columns 2 (1–3) 0 (0–2)CTB shell 2 (1–4) 0 (0–2)Interstitial trophoblast cells 2 (1–4) 0 (0–2)

Maternal:Decidual cells 3 (2–4) 0 (0–1)Fibroblast cells 4 (2–4) 0 (0–2)Glandular epithelium 1 (0–2) 0 (0–1)Venular VEC 1 (0–3) 0 (0–1Spiral arterioleb 1 (0–3) 0 (0–1)

*n 5 4. aMedian values with ranges based on 5-scale subjective scoringmethod as described in text.bBoth VEC and VSM. CTB5 cytotrophoblast,STB 5 syncytiotrophoblast, VEC5 vascular endothelial cells.

steroid hormones in a target tissue provides an understanding ofthe involvement of steroid hormone in the given tissue. If true,our observation that immunodetectable PR was present inplacental tissue during days 13–22 of gestation in monkeys,and the earlier report that PR was present in all trophoblastderived cells in human samples collected during 5–12 weeksof pregnancy (Wanget al., 1996), should mean that involvementof progesterone is required for the development and the functionof placenta in these species. Indirect evidence indicating thatvarious placental functions are indeed regulated by progester-one (Bischofet al., 1986; Das and Catt, 1987; Sitruk-Wareet al., 1990; Edwards and Brody, 1995) also supports thissuggestion. Furthermore, the observations that transformed, aswell as untransformed stromal cells in all zones of maternalendometrium, and epithelial cells of deep glands, and venularVEC and arteriolar VSM at implantation site were PR positivesuggest that endometrial–placental interaction is regulated byprogesterone. Similar observations were reported by others

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using 5–12 week pregnancy human decidua (Salmiet al.,1996; Wanget al., 1996). On the other hand, lack of PR inembryo proper, and that of ER in both embryonic, extraem-bryonic, as well as, maternal endometrial compartments inmacaque samples as observed in the present study, and inhuman samples (Salmiet al., 1996; Wanget al., 1996) maysuggest that progesterone is not essential for embryo growthand that oestradiol does not play any role in the process ofimplantation, placentation and in embryo growth in the primate.This is supported by the earlier observation that luteal phaseoestradiol-17βis not required for implantation and maintenanceof pregnancy in monkeys and women (Ghoshet al., 1994;Zegers-Hoshchild and Altieri, 1995).

Steroid hormone induced growth of target tissue is mediatedin part by the local production of polypeptide growth factors,which in turn may act in an autocrine–paracrine fashion(Zajowoski et al., 1988; Murphy and Ballejo, 1994; Seppalaand Rutanen, 1994). Thus, PR identified in trophoblast cellpopulations during early pregnancy may play a role in growthfactor mediated cell growth and differentiation. For example,the placenta is a rich source of angiogenic growth factors andtheir receptors (Charnock-Joneset al., 1994; Ahmedet al.,1995), which are believed to control trophoblast function aswell as vascular changes during implantation and placentation.The presence of PR in endometrial endothelial cells of pre-villous and villous stages of implantation supports an increasingawareness about the role envisaged for steroid hormones insupporting angiogenesis necessary for normal placentation.Wang et al. (1996) also observed PR in endothelial cellsof human first trimester pregnancy samples. Microvascularendothelial cells of tumours have also been shown to expressPR (Khalidet al., 1997). Distinctions in cell shape and cellularphysiology may be associated with the reported failure todetect steroid hormone receptors in endothelial cells of non-conception cycles (Bergeronet al., 1988; Lesseyet al.,1988), since at implantation and early stages of placentationendothelial cells acquire a hypertrophied columnar appearance(Enderset al., 1985).

PR-positive stromal–decidual cells adjacent to plaque aciniand to dilated venules at fetal–maternal junctions, and totrophoblast cells may play a critical role in regulating cell andmatrix reorganization during implantation and early pla-centation in the rhesus monkey. In human endometrial explantsin culture, progesterone at physiological concentrations hasbeen shown to inhibit the expression of matrix metalloproteases(MMP) (Marbaix et al., 1992). Secretion of tissue inhibitorof metalloprotease-1 (TIMP-1) occurs through progesteronemediated up-regulation of transforming growth factor-β (TGF-β) by human decidual cells and trophoblast cells (Graham andLala, 1992; Irving and Lala, 1995).

However, many issues are yet to be addressed:(i) The physiological significance of the observation that

epithelial cells of deeper glands were PR positive, while glandsin superficial and principal zones were conspicuously PRnegative is not known. The glandular epithelial stem cellslocated in basal zone respond to a particular micro-environmentand are self-maintaining and can repopulate, while epithelialcells in upper functionalis may not, despite a similar systemic

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milieu of the steroid hormones (Padykulaet al., 1984; Ferenczy,1994). Based on earlier evidence (Amsoet al., 1994) and theresults of the present study, it is suggested that alterations insteroid receptors may be one component of an intracellularregulatory mechanism which influences the cell cycle towardsglandular hyperplasia in pregnancy cycles (Hertig, 1964; Ghoshet al., 1993). Further studies are required to test this suggestion.

(ii) The physiological significance of relatively less intenseimmunodetectibility of PR and vimentin in decidual cells,especially in implantation sites, is not known. It has beensuggested that trophoblast cell invasion affects the decidualresponse of stromal fibroblasts (Wuet al., 1990; Shiet al.,1993; Wanget al., 1996). The nature of the trophoblast signaland the physiological relevance of such changes are open toinvestigation.

(iii) Moreover, we have no understanding about the physio-logical significance and mechanism of the regulation of PRexpression in trophoblast derived cells, especially in view ofthe fact that it is higher in villous stage tissue compared withpre-villous stage tissue, and that its expression is lower inCTB column cells as compared to that in floating villi. CTBcells in floating villi exist as a polarized epithelial monolayeranchored to the basement membrane, while CTB cells inmultilayer column are non-polarized with a non-polarized actincytoskeleton, and show associated changes in the repertoire ofintegrin receptors, which is thought to be crucial for theacquisition of its invasive phenotype (Damskyet al., 1992;Thie et al., 1997) through the selective secretion of MMPnecessary for digestion of the extracellular matrix and migrationof CTB cells (Bischof and Campana, 1996). Whether differen-tial distribution of PR in non-polarized CTB cells comparedwith that in polarized CTB cells is involved in regulating theseevents remains to be examined.

(iv) It has been demonstrated by other groups that microwavestabilization enhances immunohistochemical detection of ERin the macaque oviduct (Slaydenet al., 1995), and thatmicrowave retrieval and trypsinization enhance the immunode-tectibility of ER and PR in fixed, paraffin embedded tissuesamples (Shiet al., 1991; Szekereset al., 1994a,b). Inthe present study, it was revealed that immunohistochemicalstaining using a method in which microwave retrieval wasfollowed by trypsinization was better for both ER and PRcompared with that in which trypsinization was done beforemicrowave retrieval. However, as noted by others, such treat-ment may enhance endogenous avidin-binding activity andperoxidase activity (Wood and Warnke, 1981; Finley andPerutz, 1982; Fisheret al., 1997). In the present study,we observed that tissue peroxidase activity after microwaveretrieval and trypsinization contributed significantly to cyto-plasmic non-specificity, especially in the fetal placental cells.In the present study, immunodetectable steroid receptors weremainly nuclear when endogenous peroxidase like activityor avidin-binding activity was quenched. Nevertheless, anoccasional cytoplasmic staining was also observed in interstitialand intravascular trophoblast cells, and endothelial cells ofvenules at implantation sites. In an earlier study, Wanget al.(1996) observed a significant degree of cytoplasmic stainingin STB and CTB cells in human first trimester placental tissue

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samples. Indeed, various groups have located steroid hormonereceptors in nuclear and cytoplasmic areas of hormone-respons-ive cells of normal tissues and invasive carcinomas (Perrot-Applanatet al., 1986; Andersen, 1992; Donegan, 1992; Creas-man, 1993; Guiochon-Mantel and Milgrom, 1993). Furtherstudies are required to decipher the biological significance ofcytoplasmic steroid receptors in target cells during placentationin primates.

(v) Finally, the shape of the implantation site of the rhesusmonkey is bi-discoid with a primary implantation site at theembryonic pole and a secondary implantation site at theabembryonic pole (Ramsey, 1982). In the present study, thedistribution of immunopositive ER and PR were examinedonly at the primary implantation site. Thus, it would beinteresting to examine the distribution profiles of ER and PRfurther in the secondary implantation site.

AcknowledgementThe research study was funded by grants from the RockefellerFoundation, USA, and the Department of Biotechnology, Govt ofIndia, the Council of Scientific and Industrial Research, India. Weacknowledge the assistance provided by the Special Programme ofResearch, Development and Research Training in Human Reproduc-tion, World Health Organization, Geneva, in supplying to us thesteroid RIA reagents.

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Received on June 1, 1998; accepted on November 9, 1998

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