Immunohistochemical localization of receptors for progesterone and oestradiol-17ß in the implantation site of the rhesus monkey

Debabrata Ghosh, Surajit Dhara, Arvind Kumar and Jayasree Sengupta1

Department of Physiology, All India Institute of Medical Sciences, New Delhi 110029, India


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of the present study was to examine the cellular basis of the involvement of oestradiol and progesterone in blastocyst implantation in the primate. To this end, the cellular distribution of receptors for oestradiol (ER) and progesterone (PR) in fetal trophoblast cells and in endometrial compartments of timed lacunar (pre-villous) and villous stages of placentation in primary implantation sites collected on days 13–22 of gestation were investigated in rhesus monkeys. Both in pre-villous stage tissue and in villous stage tissue, cytotrophoblast cells and syncytiotrophoblast cells and other trophoblast derived cells were PR positive, while they were generally ER negative. Maternal endometrial cells were ER negative, while epithelial cells, stromal cells and vascular endothelial cells in maternal endometrium showed heterogeneous staining patterns for PR depending on their relative location; these patterns, however, correlated well with glandular hyperplasia and differentiation, stromal–decidual transformation and vascular response seen during blastocyst implantation.

Key words: cytotrophoblast /endometrium/oestrogen receptor/progesterone receptor/syncytiotrophoblast


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Macroscopic and microscopic characteristics of implantation and placentation stages in humans have been described by Hertig and Rock (1944) and in the macaque by Wislocki and Streeter (1938). The ultrastructural features of initial penetration by trophoblast cells of maternal tissues (Renius et al., 1973Go; Enderset al., 1983Go; Enders and King, 1991Go), formation and differentiation of extraembryonic mesoderm (Luckett, 1978Go; Enders and King, 1988Go) and the nature of endometrial responses to trophoblast penetration (Enders et al., 1985Go) in the macaque have also been described. However, the endocrine equivalents of trophoblast invasion and placentation in a stage-specific manner are not known for the rhesus monkey in particular, and for primates in general. Elevated concentrations of oestrogen, progesterone and the presence of chorionic gonadotrophin and relaxin in the systemic circulation are the associated endocrine features of conception cycles in women and in macaques (Seki et al., 1985Go; Stewart et al., 1993Go; Roberts and Anthony, 1994Go; Ghosh et al., 1997Go). There is evidence to suggest that progesterone is essential for supporting implantation associated events in the uterus, while oestradiol from ovarian sources may not be required for implantation in non-human primates and women (Ghosh et al., 1994Go; Zegers-Hoshchild and Altieri, 1995Go). However, oestradiol from embryonic and endometrial sources (Tseng et al., 1986Go; Edgar et al., 1993Go) may still play a permissive role in embryo implantation (Ghosh and Sengupta, 1995Go). To understand the cellular basis of hormone action, it is useful to investigate the cellular distribution of receptors for steroid hormones. The use of monoclonal antibodies against receptors for oestradiol (ER) and progesterone (PR) permits cell-type specific localization of steroid receptors (Press et al., 1984Go; Press and Greene, 1988Go). Through such studies, it has been reported that ER and PR show cyclical variations in endometrial glandular and stromal cells during the menstrual cycle (McClellan et al., 1984Go; Press et al., 1984Go; Lessey et al., 1988Go; Press and Greene, 1988Go; Amso et al., 1994Go). In the present study the immunolocalization of ER and PR in fetal trophoblast cells and in cells of the maternal endometrial compartment during lacunar and villous stages of placentation in the rhesus monkey was investigated.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
The details of animal management are given elsewhere (Ghosh et al., 1993Go, 1996Go, 1997Go). Briefly, proven fertile male and female rhesus monkeys were housed singly under semi-natural conditions in the Primate Research Facility of the All-India Institute of Medical Sciences, and were fed with regular monkey pellet diet, semi-formulated Indian bread and fresh seasonal fruits, and water ad libitum. Females were allowed to cohabit during days 8–16 of their menstrual cycles with males, and during this time twice daily peripheral blood samples were collected from female animals for the assessment of oestradiol-17ß, progesterone and chorionic gonadotrophin (CG) concentrations in peripheral circulation using radioimmunoassay techniques in order to detect the day of ovulation (day 0) as described previously (Ghosh et al., 1993Go, 1996Go, 1997Go). Vaginal smears were checked daily for the presence of spermatozoa. The experimental design of the present study was approved by the Ethics Committee on the Use of Non-Human Primates in Biomedical Research of the All-India Institute of Medical Sciences.

Tissue collection and processing
Prediction of pregnancy was made on the basis of elevated profiles of oestradiol-17ß and progesterone and detectable CG in peripheral circulation (Ghosh et al., 1997Go). On estimated days 13–22 (n = 14) post-fertilization, animals were laparotomized under ketamine (12 mg/kg body weight; Parke Davis & Co., Mumbai, India) anaesthesia and after checking for the presence of functional corpus luteum, in-situ perfusion was performed using sterile phosphate buffered saline (pH 7.4) followed by freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. In-situ perfusion fixation was performed for 45–60 min, then the uterus was quickly excised and placed in fresh fixative on ice for transportation to the laboratory. In the laboratory, the uterus was carefully opened to expose its luminal surface, the primary implant site was located, and carefully excised and kept for fixation for 24 h at 4°C and then processed in a routine manner for paraffin embedding (Ghosh et al., 1993Go, 1996Go, 1998Go).

Analysis of implantation sites and endometrial compartments
Based on light microscopic examination of haematoxylin stained paraffin sections, the implantation stages for each sample were documented based on earlier descriptions (Hendrickx and Houston, 1976Go; Houston, 1976Go; Enders, 1993Go). Implantation sites and associated fetal and maternal compartments were analysed using the zonation description given by Enders and King (1991). According to this description, these are: superficial zone, principal zone, transitional zone and basal zone. The superficial zone includes luminal epithelium and subepithelial compartment containing first part of glands and stroma, and implanted embryo. The principal zone forms the major part of endometrium, and is occupied by the straight part of glands, and in the narrow transitional zone by coiled glands. Basal zone contains glands characterized by tall columnar epithelia. Immunohistochemical localization for cytokeratin, vimentin, and von Willebrand factor (vWf) was performed on parallel sections to distinguish trophoblast cells, mesenchymal–stromal–decidual cells and endothelial cells respectively at implantation sites.

Immunohistochemistry
Paraffin blocks were serially sectioned at 5 µm using a Supercut microtome (Leica, Germany) and sections were collected on poly-l-lysine precoated glass slides. Immunohistochemical localization for ER and PR was performed using mouse monoclonal antisera against ER and PR (Immunotech, Cedex, France) respectively. ER-specific monoclonal antibody, ER/D5 was produced by using spleen cells from BALB/c mice immunized with recombinant ER protein, and PR-specific monoclonal antibody, PR10A9 was produced against a synthetic peptide that contains the carboxy-terminal 12 amino acids of PR. Immunodetection of ER and PR were performed using a microwave method of antigen retrieval (three heating cycles of 5 min each in 0.1 M citrate buffer, pH 6.0) and trypsinization (0.05%, w/v for 10 min) as described by Szekeres et al. (1994a,b) and Nayak et al. (1998). For both ER and PR, two methods (i.e. microwave retrieval followed by trypsinization, and trypsinization followed by microwave retrieval) were performed on serial sections. Additionally, we examined whether protein unmasking by the above retrieval treatments could result in enhanced tissue peroxidase activity and biotin activity, since a biotin–avidin–peroxidase method was employed for final visualization. In order to examine the peroxidase activity, following retrieval treatment, the sections were post-treated with hydrogen peroxide (0.3%, v/v) in phosphate buffered saline (pH 7.4) and used for PR and ER immunohistochemistry (Finley and Perutz, 1982Go). To examine the avidin-binding activity, sections were treated with avidin (0.01%, w/v) followed by biotin (0.001%, w/v) as described by Fisher et al. (1997) and subjected to immunohistochemistry for ER and PR. Sections were incubated in primary antibody overnight at 4°C. This was followed by incubation with biotinylated secondary antibody and final visualization was achieved by using the ABC peroxidase kit (Vector Laboratories, Burlingame, CA, USA) and freshly prepared 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide according to the protocol provided by the manufacturer. Since immunopositive stainings for ER and PR were detected primarily in nuclear compartment and occasionally in cytoplasm, no counter staining was done in these sections.

It was observed that staining was relatively discrete and more intense with the method of microwave retrieval followed by trypsinization for both ER and PR compared with the staining performed using a method in which microwave retrieval in citrate buffer was done after trypsinization. Incubation with hydrogen peroxide following microwave 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 activity exposed by retrieval treatment failed to reduce any further non-specificity in the staining pattern. Thus, sections subjected to microwave retrieval treatment in citrate buffer followed by trypsinization, post-treated for quenching of peroxidase and avidin-binding activities, and finally immunohistochemically stained with respective monoclonal antibodies for ER and PR were analysed for their immunopositive distribution in different fetal and maternal compartments during pre-villous and villous stages of placentation.

As mentioned above, parallel sections of implantation sites from each sample were processed for immunocytochemical localization of cytokeratin, vimentin and vWf using specific monoclonal antisera (Dako-CK MNF116, Dako-vimentin V9, and Dako-vWf F8/86 respectively) obtained from Dako A/S (Glostrup, Denmark) and final visualization was obtained using the ABC peroxidase kit and freshly prepared diaminobenzidine hydrochloride with hydrogen peroxide as described above. These sections were counter-stained with haematoxylin.

Dilutions of stock primary antibodies for incubation were precalibrated based on 3–5 points titration and the information provided by the manufacturers. Specificity of antibody liganding and visualization were assessed by (i) omitting primary antibodies, (ii) replacing primary antibodies with unrelated immunoglobulins from same species and other species, (iii) omitting secondary antibodies, and (iv) replacing labelled secondary antibody with unrelated labelled immunoglobulins from same species and other species. For a given probe, all sections were subjected to immunohistochemistry simultaneously. Late proliferative and early luteal phase endometrial tissue sections were used as positive controls. Labelled and unlabelled immunoglobulins, non-immune sera, and visualization kits (horseradish peroxidase) were purchased from Vector Laboratories. All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA).

For assessment of immunostaining in cells of embryonic and endometrial compartments, semiquantitative subjective scoring was done based on a 5-scale system: 0, nil (0%); 1, very weak (< 5%); 2, weak (5–25%); 3, moderate (25–75%); 4, strong (> 75%), as described by Press et al. (1988). It was assumed that these measurements reflect the concentrations of the experimental protein in different fetal and endometrial compartments.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Table IGo gives the detailed description of samples investigated in the present study. Figure 1Go shows representative lacunar (pre-villous) and villous stages as obtained in the present study. Table IIGo shows the immunohistochemical distribution of cytokeratin and vimentin in pre-villous and villous stage implantation sites on days 13–22 of gestation. Immunolocalization revealed cytokeratin-positive staining in all populations of trophoblast cells (Figure 2a–dGo). Intense immunopositive staining for cytokeratin was observed in syncytiotrophoblast (STB) cells lining lacunae and villi, and moderate staining for cytotrophoblast (CTB) cells present as non-polarized columns of cells. Venules and spiral arterioles in principal zone were often infiltrated with cytokeratin-positive trophoblast cells; interstitial trophoblast cells were also cytokeratin-positive (Figure 2cGo). Extraembryonic mesenchymal cells were vimentin-positive (Figure 2e, fGo), villous mesenchymal cells were also discretely cytokeratin-positive (Figure 2dGo). Vimentin-positive endometrial stromal cells surrounding plaque acini and adjoining dilated blood vessels (Figure 2g,hGo) were seen. Within the implantation site, vimentin-positive stromal cells were often detected as discrete groups of cells at the lower end of the trophoblast plate (Figure 2g, hGo). Vimentin-positive stromal cells were also observed in the superficial zone on both sides of the implant. Decidual cells adjacent to the CTB shell were positive for vimentin. At this stage of placental development, blood vessels – mainly venules – were dilated, and vWf-positive vascular endothelial cells (VEC) were markedly hypertrophied at implantation sites (Figure 2iGo).


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Table I. Description of implantation sites recovered from rhesus monkeys
 


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Figure 1. Representative micrographs of haematoxylin stained, paraffin sections of implantation sites obtained from: (A) animal 2459 on estimated day 13 of gestation. Bilaminar embryonic disc (ED), cytotrophoblast and syncytiotrophoblast cells forming an expanded trophoblast plate (TP), small clefts and trophoblastic lacunae (L) are seen in this region. The uterine epithelium is completely missing over the area of contact between embryo and endometrium. A dilated blood vessel (BV) continuous with trophoblast lacunae immediately below the implantation site, maternal red blood cells can be seen within lacunae. At higher magnification (a), ED is characterized by loosely arranged cells of epiblast (ep) and endoderm (end). (B) Animal 2444 on estimated day 14 of gestation. Well defined bilaminar embryonic disc (ED), intervillous spaces (IVS) and plaque acini (PL) are seen. In higher magnification (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 on estimated day 16 of gestation. The cells of epiblast have differentiated 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 is elevated from uterine surface and protrudes into uterine cavity. A thin membraneous chorion (CH) with an inner mesothelial layer extends from edge of placenta into uterine cavity. Cytotrophoblast cell columns (CC) from the tips of open villi to the base of placenta are distinct. Numerous dilated BV are continuous with intervillous spaces (IVS) which are filled with maternal blood. (D) Animal 2318 on estimated day 22 of gestation. Distinctly branched chorionic villi are seen. Thick cytotrophoblastic shell (SH), darkly stained junctional zone (JCT) containing trophoblast cells, necrotic cells and decidual cells, an area of decidua basalis (DB) are seen. Bars = 300 µm (A, B, C, D) and 1000 µm (a, b).

 

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


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Figure 2. Micrographs of immunohistochemically stained sections for cytokeratin, vimentin and vWf. (a) Immunopositive cytokeratin staining 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. Bar = 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 migratory column of cells at the fetal–maternal border of implantation site (X), interstitial trophoblast cells (arrows) and plaque cells (arrow heads) are cytokeratin-positive. Bar = 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 cells present within maternal stroma (arrows), and within maternal blood vessel (arrow head). Bar = 50 µm. (d) Secondary villi showing immunostaining for cytokeratin. Syncytiotrophoblast cells (STB) and cytotrophoblast cells (CTB) lining villi show positive staining. Note the delicately positive stain in extraembryonic mesodermal cells (arrows). Bar = 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. Bar = 50 µm. (f) Immunohistochemical staining for vimentin of implantation site. Extraembryonic mesodermal cells (arrows) adjoining primary villi and within secondary villi are postively stained. Bar = 50 µm. (g) Vimentin immunostaining in maternal stromal cells of implantation site. Implantation zone lying below lacunae (L) shows that trophoblast cells are negative, while stromal cells stain intensely for vimentin. Bar = 50 µm. (h) Vimentin immunostaining of cells adjoining implantation site. Plaque cells (PL), luminal epithelia (LE) are negative, stromal cells located within and surrounding plaque acini and blood vessel are strongly positive. Bar = 80 µm. (i) von Willebrand factor immunostaining of endothelial cells (arrows) of dilated blood vessels at fetal–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. Bar = 80 µm.

 
Figures 3–5GoGoGo show immunolocalization of PR and ER in different cell types of embryonic and endometrial compartments during pre-villous and villous placentation. Tables III and IVGoGo provide the scores of immunopositive PR in cells of fetal and endometrial compartments at implantation sites and endometrial cells adjacent to implantation sites, respectively in 10 tissue samples collected during days 13–17 of gestation. On the fetal side, CTB cells lining embryonic cavity, CTB and STB cells lining lacunae and villi, and trophoblast cells in trophoblast plate were PR positive (Table IIGo; Figures 3a, 4aGoGo). Polarized CTB cells associated with lacunae and villi were generally more intensely PR positive compared with STB cells and non-polarized CTB cells of the column. On the endometrial side, only stromal cells in all zones, and glandular epithelial cells in transitional and basal zones, and few venular VECs at implant sites, and vascular smooth muscle (VSM) of spiral arterioles in superficial and principal zones, were characteristically PR positive (Tables III and IVGoGo; Figures 4b, 5GoGo). Generally, decidual cells at implantation sites showed less intense PR positivity compared with stromal cells in associated areas. Some intravascular trophoblast cells showed cytoplasmic staining (Figure 4bGo). Since there was little immunodetectable ER cell types in the samples of gestation days 13–17, data are not shown (see Figure 3cGo). Table VGo shows the scores of immunopositive PR and ER in cells of fetal and endometrial compartments at implantation sites from four samples collected on day 22 of gestation. In these samples, the overall discernibility of positive signals was generally higher compared with earlier stages, however, there was no change in the basic pattern of immunostaining for ER and PR in fetal and endometrial sides; CTB and STB cells surrounding villi (Fig. 4cGo), interstitial trophoblast cells and trophoblast cells in CTB shell (Figure 3bGo), intravascular trophoblast cells (Figure 4dGo), and stromal cells in endometrium showed PR positivity. Except for occasional trophoblast cells (Figure 3dGo), cells in both compartments were ER negative.



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

 


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Figure 4. PR immunostaining in trophoblast cells lining villi, and cytotrophoblast cell column (CC), and extraembryonic mesodermal cells (arrows ) of implantation site recovered on day 16 of gestation (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 with cytotrophoblast cells (CTB) showing stronger immunostaining compared with syncytiotrophoblast cells (STB), and non-polarized cytotrophoblast cells of cell column (CC) showing weak and infrequent staining (c), in trophoblast cells (*) within maternal blood vessel in endometrium at 22 days of gestation (d). Note the positive immunostaining in endothelial cells (arrow) (d). Bars = 45 µm (a), 30 µm (b, c) and 20 µm (d).

 


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Figure 5. PR immunostaining of implantation site associated endometrium. Stromal cells in oedematous area adjacent to the implant 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 epithelial cells (GE) express heterogeneity in staining for PR; stromal cells staining for PR were less intensely stained than that in the principal zone (c). Glandular epithelial cells (GE) and stromal cells in basalis zone are positively stained (d). Bar = 45 µm.

 

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

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Table IV. Immunohistochemical localization of progesterone receptors in endometrial cells of pre-villous and villous stage implantation sites on days 13–17 of gestation*
 

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

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The light microscopic examination of primary implantation sites during early stages (days 13–22) of pregnancy of the rhesus monkey revealed the following observations. In samples of day 13 of gestation, lacunae of different dimensions were evident in trophoblast plates. Distinctive endometrial responses to invading trophoblasts, which included subepithelial oedema, and epithelial plaque cells adjacent to implantation sites were evident. On subsequent days, the expansion of lacunae was followed by formation of primary villi (by days 14–15 of gestation) and then secondary villi (days 15–17) and tertiary villi (day 22), and was associated with substantial expansion of implantation sites. At this time, oedema was reduced, regressive changes in plaque cells became distinctive, and stromal cell recruitment to decidual transformation was enhanced. Throughout this period, venular dilatation, blood vessel penetration by trophoblast cells, and vWf-positive endothelial cell hypertrophy were remarkable features at implantation sites. These observations are very similar to those reported earlier in the same species (Wisloki and Streeter, 1938; Heuser and Streeter, 1941Go; Enders et al., 1983Go, 1985Go; Enders and Schlafke, 1986Go; Enders, 1993Go). Immunolocalization of cytokeratin and vimentin in these samples revealed that all trophoblast cells were cytokeratin-positive including STB and CTB lining lacunae and villi, interstitial trophoblast cells and endovascular cytotrophoblast cells (EVCT), while mesenchymal cells of the amniochorion and chorionic villi were positive for both vimentin and cytokeratin. Similar observation has been reported earlier in human samples collected in week 36 of gestation (Khong et al., 1986Go) and in monkey samples collected on days 22–160 of gestation (Blankenship et al., 1993Go). It is to be noted that the purpose of immunostaining for cytokeratin, vimentin and vWf was to identify trophoblast cell, mesenchymal cell and endothelial cell populations in maternal endometrial stromal compartment, especially at loci where these cell types co-exist in close juxtaposition and that we made no attempt to examine the specificity of cytokeratin-type expressed by different subsets of trophoblasts according to location and stage of differentiation. Indeed, there is evidence that differentiation of human trophoblast cell populations involve alterations in the patterns of cytokeratin expression (Muhlhauser et al., 1995Go; Vicovac and Aplin, 1996Go).

We now demonstrate for the first time the pattern of distribution 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 generally believed that knowledge of cellular distribution of receptors for steroid hormones in a target tissue provides an understanding of the involvement of steroid hormone in the given tissue. If true, our observation that immunodetectable PR was present in placental tissue during days 13–22 of gestation in monkeys, and the earlier report that PR was present in all trophoblast derived cells in human samples collected during 5–12 weeks of pregnancy (Wang et al., 1996Go), should mean that involvement of progesterone is required for the development and the function of placenta in these species. Indirect evidence indicating that various placental functions are indeed regulated by progesterone (Bischof et al., 1986Go; Das and Catt, 1987Go; Sitruk-Ware et al., 1990Go; Edwards and Brody, 1995Go) also supports this suggestion. Furthermore, the observations that transformed, as well as untransformed stromal cells in all zones of maternal endometrium, and epithelial cells of deep glands, and venular VEC and arteriolar VSM at implantation site were PR positive suggest that endometrial–placental interaction is regulated by progesterone. Similar observations were reported by others using 5–12 week pregnancy human decidua (Salmi et al., 1996Go; Wang et al., 1996Go). On the other hand, lack of PR in embryo proper, and that of ER in both embryonic, extraembryonic, as well as, maternal endometrial compartments in macaque samples as observed in the present study, and in human samples (Salmi et al., 1996Go; Wang et al., 1996Go) may suggest that progesterone is not essential for embryo growth and that oestradiol does not play any role in the process of implantation, placentation and in embryo growth in the primate. This is supported by the earlier observation that luteal phase oestradiol-17ß is not required for implantation and maintenance of pregnancy in monkeys and women (Ghosh et al., 1994Go; Zegers-Hoshchild and Altieri, 1995Go).

Steroid hormone induced growth of target tissue is mediated in part by the local production of polypeptide growth factors, which in turn may act in an autocrine–paracrine fashion (Zajowoski et al., 1988Go; Murphy and Ballejo, 1994Go; Seppala and Rutanen, 1994Go). Thus, PR identified in trophoblast cell populations during early pregnancy may play a role in growth factor mediated cell growth and differentiation. For example, the placenta is a rich source of angiogenic growth factors and their receptors (Charnock-Jones et al., 1994Go; Ahmed et al., 1995Go), which are believed to control trophoblast function as well 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 increasing awareness about the role envisaged for steroid hormones in supporting angiogenesis necessary for normal placentation. Wang et al. (1996) also observed PR in endothelial cells of human first trimester pregnancy samples. Microvascular endothelial cells of tumours have also been shown to express PR (Khalid et al., 1997Go). Distinctions in cell shape and cellular physiology may be associated with the reported failure to detect steroid hormone receptors in endothelial cells of non-conception cycles (Bergeron et al., 1988Go; Lessey et al., 1988Go), since at implantation and early stages of placentation endothelial cells acquire a hypertrophied columnar appearance (Enders et al., 1985Go).

PR-positive stromal–decidual cells adjacent to plaque acini and to dilated venules at fetal–maternal junctions, and to trophoblast cells may play a critical role in regulating cell and matrix reorganization during implantation and early placentation in the rhesus monkey. In human endometrial explants in culture, progesterone at physiological concentrations has been shown to inhibit the expression of matrix metalloproteases (MMP) (Marbaix et al., 1992Go). Secretion of tissue inhibitor of metalloprotease-1 (TIMP-1) occurs through progesterone mediated up-regulation of transforming growth factor-ß (TGF-ß) by human decidual cells and trophoblast cells (Graham and Lala, 1992Go; Irving and Lala, 1995Go).

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 glands in superficial and principal zones were conspicuously PR negative is not known. The glandular epithelial stem cells located in basal zone respond to a particular micro-environment and are self-maintaining and can repopulate, while epithelial cells in upper functionalis may not, despite a similar systemic milieu of the steroid hormones (Padykula et al., 1984Go; Ferenczy, 1994Go). Based on earlier evidence (Amso et al., 1994Go) and the results of the present study, it is suggested that alterations in steroid receptors may be one component of an intracellular regulatory mechanism which influences the cell cycle towards glandular hyperplasia in pregnancy cycles (Hertig, 1964Go; Ghosh et al., 1993Go). Further studies are required to test this suggestion.
(ii) The physiological significance of relatively less intense immunodetectibility of PR and vimentin in decidual cells, especially in implantation sites, is not known. It has been suggested that trophoblast cell invasion affects the decidual response of stromal fibroblasts (Wu et al., 1990Go; Shi et al., 1993Go; Wang et al., 1996Go). The nature of the trophoblast signal and the physiological relevance of such changes are open to investigation.
(iii) Moreover, we have no understanding about the physiological significance and mechanism of the regulation of PR expression in trophoblast derived cells, especially in view of the fact that it is higher in villous stage tissue compared with pre-villous stage tissue, and that its expression is lower in CTB column cells as compared to that in floating villi. CTB cells in floating villi exist as a polarized epithelial monolayer anchored to the basement membrane, while CTB cells in multilayer column are non-polarized with a non-polarized actin cytoskeleton, and show associated changes in the repertoire of integrin receptors, which is thought to be crucial for the acquisition of its invasive phenotype (Damsky et al., 1992Go; Thie et al., 1997Go) through the selective secretion of MMP necessary for digestion of the extracellular matrix and migration of CTB cells (Bischof and Campana, 1996Go). Whether differential distribution of PR in non-polarized CTB cells compared with that in polarized CTB cells is involved in regulating these events remains to be examined.
(iv) It has been demonstrated by other groups that microwave stabilization enhances immunohistochemical detection of ER in the macaque oviduct (Slayden et al., 1995Go), and that microwave retrieval and trypsinization enhance the immunodetectibility of ER and PR in fixed, paraffin embedded tissue samples (Shi et al., 1991Go; Szekeres et al., 1994aGo,bGo). In the present study, it was revealed that immunohistochemical staining using a method in which microwave retrieval was followed by trypsinization was better for both ER and PR compared with that in which trypsinization was done before microwave retrieval. However, as noted by others, such treatment may enhance endogenous avidin-binding activity and peroxidase activity (Wood and Warnke, 1981Go; Finley and Perutz, 1982Go; Fisher et al., 1997Go). In the present study, we observed that tissue peroxidase activity after microwave retrieval and trypsinization contributed significantly to cytoplasmic non-specificity, especially in the fetal placental cells. In the present study, immunodetectable steroid receptors were mainly nuclear when endogenous peroxidase like activity or avidin-binding activity was quenched. Nevertheless, an occasional cytoplasmic staining was also observed in interstitial and intravascular trophoblast cells, and endothelial cells of venules at implantation sites. In an earlier study, Wang et al. (1996) observed a significant degree of cytoplasmic staining in STB and CTB cells in human first trimester placental tissue samples. Indeed, various groups have located steroid hormone receptors in nuclear and cytoplasmic areas of hormone-responsive cells of normal tissues and invasive carcinomas (Perrot-Applanat et al., 1986Go; Andersen, 1992Go; Donegan, 1992Go; Creasman, 1993Go; Guiochon-Mantel and Milgrom, 1993Go). Further studies are required to decipher the biological significance of cytoplasmic steroid receptors in target cells during placentation in primates.
(v) Finally, the shape of the implantation site of the rhesus monkey is bi-discoid with a primary implantation site at the embryonic pole and a secondary implantation site at the abembryonic pole (Ramsey, 1982Go). In the present study, the distribution of immunopositive ER and PR were examined only at the primary implantation site. Thus, it would be interesting to examine the distribution profiles of ER and PR further in the secondary implantation site.


    Acknowledgments
 
The research study was funded by grants from the Rockefeller Foundation, USA, and the Department of Biotechnology, Govt of India, the Council of Scientific and Industrial Research, India. We acknowledge the assistance provided by the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization, Geneva, in supplying to us the steroid RIA reagents.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 1, 1998; accepted on November 9, 1998.