Department of Physiology, All India Institute of Medical Sciences, New Delhi 110029, India
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Abstract |
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Key words: cytotrophoblast /endometrium/oestrogen receptor/progesterone receptor/syncytiotrophoblast
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Introduction |
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Materials and methods |
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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., 1997). On estimated days 1322 (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 4560 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., 1993
, 1996
, 1998
).
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, 1976; Houston, 1976
; Enders, 1993
). 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, mesenchymalstromaldecidual 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 biotinavidinperoxidase 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, 1982). 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 35 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 (525%); 3, moderate (2575%); 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.
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Results |
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Discussion |
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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 1322 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 1322 of gestation in monkeys, and the earlier report that PR was present in all trophoblast derived cells in human samples collected during 512 weeks of pregnancy (Wang et al., 1996), 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., 1986
; Das and Catt, 1987
; Sitruk-Ware et al., 1990
; Edwards and Brody, 1995
) 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 endometrialplacental interaction is regulated by progesterone. Similar observations were reported by others using 512 week pregnancy human decidua (Salmi et al., 1996
; Wang et al., 1996
). 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., 1996
; Wang et al., 1996
) 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., 1994
; Zegers-Hoshchild and Altieri, 1995
).
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 autocrineparacrine fashion (Zajowoski et al., 1988; Murphy and Ballejo, 1994
; Seppala and Rutanen, 1994
). 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., 1994
; Ahmed et al., 1995
), 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., 1997
). 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., 1988
; Lessey et al., 1988
), since at implantation and early stages of placentation endothelial cells acquire a hypertrophied columnar appearance (Enders et al., 1985
).
PR-positive stromaldecidual cells adjacent to plaque acini and to dilated venules at fetalmaternal 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., 1992). 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, 1992
; Irving and Lala, 1995
).
However, many issues are yet to be addressed:
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Acknowledgments |
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Notes |
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References |
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Submitted on June 1, 1998; accepted on November 9, 1998.