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Regular Article |
in Bovine Placentomes Throughout Mid and Late Gestation and at Parturition1
a Klinik für Geburtshilfe, Gynäkologie und Andrologie der Groß- und Kleintiere mit Tierärztlicher Ambulanz,
b Institut für Veterinär-Physiologie, Arbeitsgruppe Biomathematik und Datenverarbeitung,
c Institut für Veterinär-Anatomie, -Histologie und -Embryologie, Justus-Liebig-Universität, D-35392 Giessen, Germany
d Max-Planck-Institut für experimentelle Endokrinologie, D-30625 Hannover, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER). Indirect immunoperoxidase staining methods were applied using primary monoclonal antibodies (pmAbs) directed against the C-terminus (AER311, HT277) or the N-terminus (AER314, 1D5) of the ER molecule. Both types of pmAbs identified ER in stromal cells and capillary pericytes of the maternal caruncular septae. Using pmAb 1D5, the mean percentage of ER-positive caruncular stromal cells decreased from 39.0% ± 5.9% in pregnant cows to 17.5% ± 8.3% at parturition (P = 0.011). Only pmAb recognizing the C-terminus identified ER in the caruncular epithelium, in which positive reactions were found in all cells, with the exception of areas adjacent to the chorionic plate and to major chorionic villi, where the specific signal gradually faded and occasionally disappeared. No positive reactions were observed in the fetal part of the placentomes. The expression of ER in bovine placentomes was further confirmed by the detection of ER-specific mRNA by reverse transcriptase-polymerase chain reaction and by Western blot analysis. The results suggest a role for placental estrogens as paracrine factors involved in the regulation of placental growth and differentiation.
estradiol receptor, placenta, pregnancy, steroid hormone receptors, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the bovine, placentome estrogen sulfotransferase and sulfosulfatase are expressed in close proximity to each other [8]. These enzymes catalyze opposite reactions; thus, free estrogens may be inactivated by sulfotransferase activity, whereas hydrolysis of sulfoconjugated estrogens by sulfatase may provide free, biologically active estrogens. The present study was designed to test for the hypothesis of whether placental estrogens may act as local regulatory factors. Consequently, expression of the estrogen receptor (ER) was assessed in the bovine placentome at the mRNA and protein levels. In addition, localization of ER was targeted qualitatively and quantitatively by immunohistochemistry in mid and late gestation and at parturition to identify putative target cells of placental estrogens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For immunohistochemistry, placentomes were collected from cows that were pregnant for 150, 220, 240, and 270 days, and from parturient cows (5 experimental groups each consisting of 3 animals). For sample collection from the pregnant cows, uteri were removed at slaughter immediately after the animals were stunned by bolt pistol. After opening the uterus and removing the fetus, cotyledonary arteries of up to five placentomes from the mid-region of the horn that contained the fetus were cannulated, and cotyledons were perfused with 10% neutral phosphate-buffered formaline as a fixative. After perfusion the placentomes were removed from the uteri and immersion-fixed for 24 h in the fixative. Placentomes from parturient cows were obtained from cows undergoing routine caesarean delivery for medical indications. Immediately after the removal of the calves, which showed normal development and were vital in all cases, single placentomes were abscised from the ligated caruncular stalk and were, for technical reasons, subjected to immersion fixation only. From all placentomes, wedge-shaped or trapezoid pieces encompassing the total height of the placentome were embedded in a paraffin substitute (Histo-Comp-Vogel, Giessen, Germany). These tissue samples had been used previously in parallel studies on progesterone receptor expression [9] and on proliferative activity [10].
For RNA extraction, placentomes from cows that had been pregnant for 150 (n = 2), 240 (n = 2), and 270 (n = 1) days were quick-frozen in liquid nitrogen immediately after they were removed from the uterus and stored at -80°C until analysis. Endometrial tissue of a nonpregnant cow serving as a positive control was obtained from a local abattoir and conserved in the same manner.
Selection of Primary Antibodies for Immunohistochemistry
With the following monoclonal antibodies, which are further specified in Table 1, specific immunostaining was observed in formalin-fixed calf endometrium which was used as a positive reference tissue: 1D5 (Biogenex/DCS, Hamburg, Germany) [11], AER311, AER314 (both from Neomarkers/Dunn Labortechnik, Asbach, Germany) [12], and HT277 [13, 14]. In calf endometrium, these four antibodies stained nuclei and cytoplasm of luminal and glandular epithelial cells, and the nuclei of a proportion of stromal cells. Sections from all placentomes were stained using 1D5 and AER311, and spot checks were performed using HT277 and AER314 for comparison.
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Immunohistochemical Staining Procedure
Indirect immunoperoxidase staining methods were applied using the streptavidin-biotin technique for signal enhancement following standard procedures with a slight modification for the different primary antibodies. Negative controls were set up with isotype-specific irrelevant monoclonal antibodies CBL600 (Cymbus Biotechnology Ltd., Hampshire, U.K.; to replace 1D5, AER314, and HT277) and 7TF-1F5 (Immunotech Diagnostics, Krefeld, Germany; to replace AER311). Tissue sections (about 4 µm) were mounted on silane-coated slides, deparaffinized by two 15-min changes of xylene, and rehydrated in graded ethanol. For antigen retrieval the rehydrated sections were preincubated in 10 mM citrate buffer pH 6.0 for 5 min before microwave irradiation at 800 W, 3 times for 5 min each, in preheated citrate buffer. After a 10-min cooling period the slides were rinsed in distilled water (2 changes, 2 min each) and PBS pH 7.2 (3 changes, 5 min each) followed by a treatment with 0.3% hydrogen peroxide in PBS for 20 min (HT277) or with 2.0% hydrogen peroxide in PBS for 5 min (1D5, AER311, AER314) in order to quench endogenous peroxidase activity. They were then covered with 10% inactivated horse serum in PBS to block unspecific binding sites. After draining the blocking reagent, the primary antibody was applied, and the slides were incubated for 20 h in a humid chamber at 4°C. They were then washed with PBS (3 changes, 5 min each), covered with biotinylated horse anti-mouse immunoglobulin G (IgG) antibody (Vector Laboratories, Burlingame, CA) diluted 1:200 in PBS, and incubated for 30 min at room temperature. Following draining of excess reagent and washing with PBS (3 changes, 5 min each) the sections were covered with streptavidin-peroxidase complex (Vector Laboratories) and incubated for 30 min. After washing with PBS (3 changes, 5 min each) the slides were immersed into the substrate solution consisting of 0.05% diaminobenzidine and 0.01% hydrogen peroxide in 50 mM imidazole-HCl buffer pH 7.1 for 5 min. The sections were then washed, counterstained with hematoxylin, and mounted in Kaisers glycerol-gelatin (Merck KgaA, Darmstadt, Germany).
Quantitative Determination of Immunohistochemically ER-Positive Caruncular Stromal Cells and Statistical Evaluation
For quantitative assessment of ER-positive caruncular stromal cells (CSCs), three sections of a randomly chosen placentome per cow were analyzed by the same person who was blind to the groups, after immunostaining with 1D5 as the primary antibody. To test for the effect of the localization within the tissue, sections encompassing the total high of the placentomes were divided into three zones of equal width: a superficial zone close to the chorionic plate (zone I), an intermediary zone (zone II), and a basal zone close to the caruncular stalk (zone III). In each zone the total number and the number of ER-positive CSCs were counted at a 200-fold magnification in at least two views arbitrarily chosen, and the percentage of ER-positive CSCs was calculated. If the total number of CSCs was less than 200, additional views were analyzed until more than 200 cells per zone had been registered.
The expression of ER in the caruncular stroma was set in relation to the observational group (Days 150, 220, 240, 270, and parturition) and the localization within the placentome (zones I–III). For statistical evaluation, a four-factorial partial hierarchic ANOVA (observational group [G], animal within observational group [A (G)], section within animal and observational group [S (AG)] and zone [Z]) was applied (BMDP statistical software, program BMDP8V [15]).
Reverse Transcription-Polymerase Chain Reaction
Coarse pieces of deep-frozen (-80°C) placentomal tissue were briefly placed in liquid nitrogen, quickly enveloped in sterile aluminum foil, and then reduced to small pieces by strokes with a hammer. The resulting tissue particles were then powdered under liquid nitrogen with a pestle in a mortar that was prechilled to -80°C. Approximately 150 mg of tissue powder were immersed in 3 ml of buffer RLT/ß-ME (RNeasy Midi Kit, Qiagen, Hilden, Germany) and homogenized by three 30-sec bursts using an ultra turrax T25 (IKA-Werke GmbH & Co. KG, Staufen i. Br., Germany). The subsequent silica-gel based RNA extraction was performed according to the instructions of the kit supplier.
For reverse transcription-polymerase chain reaction (RT-PCR), the GeneAmp RNA PCR Kit (Perkin Elmer, Foster City, CA) was used; 0.3 µg of total RNA was reverse transcribed at 42°C for 15 min in a total volume of 10 µl containing 2.5 µmol l-1 random hexamers, 1 mmol l-1 dNTPs, RNase inhibitor (10 units per reaction) and murine leukemia virus reverse transcriptase (25 units per reaction). The reaction was stopped by heating to 99°C for 5 min. The cDNA mix was amplified in a Personal Cycler (Biometra GmbH, Göttingen, Germany) after addition of 15 pmol primers and 0.25 units of AmpliTaq DNA Polymerase per reaction in a 50-µl reaction volume containing 1 mM l-1 MgCl2. The primers were specific to a 477 base pair (bp)-fragment spanning exons V to VII within the hormone binding domain of the bovine ER ([16], GenBank accession number U64962). In parallel, primers for bovine ß-actin [17] were used to amplify a 890 bp-fragment as a positive control. A negative control was set up using H2O instead of RNA in the RT reaction mix. The program consisted of 34 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The products were analyzed on a 2% agarose gel stained with ethidium bromide and visualized under UV transillumination.
Western Blot Analysis
Tissue powder (600 mg) from a placentome of a 150-day pregnant cow and from the endometrium of a nonpregnant cow (prepared as described above) were homogenized with an ultra turrax T25 in 3 ml of ice-cold PBS with protease inhibitor cocktail tablets (Complete Mini, Roche Diagnostics GmbH, Mannheim, Germany). SDS was added at a final concentration of 5% and the samples were boiled for 10 min. After centrifugation for 10 min at 3500 x g the protein content in the supernatant was determined using a BioPhotometer (Eppendorf AG, Hamburg, Germany). Samples (50 µg protein) were boiled in 3x loading buffer (10 mM Tris-HCl pH 6.8 including 3% SDS, 5% ß-mercaptoethanol, 20% glycerol, and 0.6% bromophenol blue) for 3 min, separated on a 10% polyacrylamide gel under reducing conditions, and transferred to nitrocellulose membranes (Optitran BA-S85, Schleicher & Schüll, Dassel, Germany). For blocking, membranes were incubated in PBS-T (PBS with 0.05% Tween-20) with 5% nonfat dry milk overnight. Then the membranes were washed in PBS-T and incubated for 75 min with the respective primary murine monoclonal anti-ER antibody: HT277, AER311, or 1D5. After washing in PBS-T, the membranes were incubated with the horseradish peroxidase-linked secondary anti-mouse immunoglobulin antibody (NA931; Amersham Pharmacia Biotech, Freiburg, Germany) for 45 min. Finally, they were again washed in PBS-T, incubated in enhanced chemiluminescence reagents (ECL Western Blotting Analysis System, Amersham Pharmacia Biotech, Freiburg, Germany) for 1 min, and exposed to VA711B Blue Sensitive x-ray films (Valmex GmbH, Augsburg, Germany), which were developed as usual for radiograms. Negative controls were set up to replace the primary monoclonal anti-ER antibodies with an isotype-specific irrelevant monoclonal antibody (CBL600) or PBS. As a positive reference sample (provided by Dr. A. Baniahmad, Genetic Institute, University of Giessen), an extract of HEK-293 cells transiently transfected with a plasmid encoding human ER was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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molecule. Both types of antibodies identified ER in the caruncular stroma, where positive nuclear reactions were found in a proportion of the fibrocyte-like cells (Fig. 1, A–C) and occasionally also in pericytes attached to capillaries (Fig. 1C). The mean percentage of ER-positive caruncular stromal cells (Fig. 2) decreased from 39.0% ± 5.9% in pregnant cows to 17.5% ± 8.3% at parturition (P = 0.011), and was higher in zone III adjacent to the caruncular stalk than in the intermediate and superficial zones II and I, respectively (P = 0.001). Visually, no changes in staining intensity were observed between Days 150 and 270, but it was clearly lower at parturition; the distinct positive staining was mainly restricted to the areas close to the caruncular stalk (Fig. 3A).
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In caruncular epithelium only the primary antibodies recognizing the C-terminus (AER311, HT277) gave positive signals (Fig. 1, B and E–H) which were predominantly nuclear, but to a lesser extent also cytoplasmic. Between Days 150 and 270, homogenous positive staining was found in all caruncular epithelial cells (Fig. 1E) with the exception of areas immediately adjacent to the chorionic plate and to primary chorionic villi (Fig. 1F), where the signal intensity became gradually weaker and occasionally disappeared entirely. These regions were disregarded when nuclear staining intensity of caruncular epithelial cells was evaluated, which varied considerably between individual animals, but showed no obvious relation to the observational group (Table 2). ER-positive reactions were still present in degenerating cells exfoliating from the caruncular epithelium into the trophoblast (Fig. 1G). Furthermore, the maternally derived nucleus of feto-maternal hybrid cells, formed of caruncular epithelial cells and weakly invasive trophoblast giant cells, exhibited positive staining, whereas the fetally derived nuclei of these hybrid cells always stained negatively (Fig. 1, B and H). In placentomes from parturient cows, distinct to intense signals were still present in caruncular epithelial remnants (Fig. 3B).
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In the negative controls using irrelevant murine monoclonal IgG antibodies instead of the specific primary antibodies, occasionally a weak to moderate diffuse staining was observed along the feto-maternal borderline, which was not nuclear in origin (Fig. 1D).
RT-PCR in placentomes from cows that were pregnant for 150–270 days (n = 5) and control endometrium using primers specific to the bovine ER yielded a DNA fragment of the expected size of 477 bp (Fig. 4). No further amplifications could be detected in any of these samples.
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By Western blot analysis of a placentome from a 150-day pregnant cow (Fig. 5), a distinct band of the expected molecular weight of approximately 67 kDa was found using the C-terminal-directed pmAb AER311 (Fig. 5A) and HT277 (Fig. 5B), albeit clearly weaker compared with the signal found in the endometrium of a nonpregnant cow, which was used as a positive reference tissue. Using the N-terminal-directed pmAb 1D5 (Fig. 5C), a weak and washy band appeared at the position corresponding to ER only after prolonged exposition of the membrane to the x-ray film, although a strong signal was again detected in the endometrium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[12]. HT277 was obtained after immunization against the porcine ER [13, 14], and 1D5 after immunization against recombinant human ER [11]. However, it was surprising that these antibodies produced different staining patterns in the bovine placentome. Although all four antibodies gave a specific nuclear staining in caruncular stromal cells, only the two antibodies directed against the C-terminus of the ER molecule recognized ER in caruncular epithelium, yielding nuclear and cytoplasmic staining. These observations suggest an interrelationship between the localization of the epitope of the respective primary antibody and its capability of binding to ER in caruncular epithelium. A possible explanation may be a differential steric hindrance induced by ligand binding or hormone status-dependent binding of the receptor to DNA or ER-associated regulatory proteins such as coactivators or corepressors [18]. Microwave irradiation, which was used for antigen retrieval, may induce breakages of fixer-induced cross-linkages. However, in porcine uterus it has been shown that the unmasking of ER with microwave treatment was not sufficient to retrieve all determinants completely, and it was concluded that the degree of accessibility of antibody epitopes differs according to the functional cell type [13]. Thus, in order to obtain unbiased results it has been recommended that we use not only one but several antibodies to different epitopes of the ER [13], which we followed in this study.
Alternatively, or in addition, in caruncular epithelium, N-terminal targeted antibodies could also be prevented from binding by hyperphosphorylation of ER, as four phosphorylation sites present in the human ER where hormone inducible phosphorylation occurs have been mapped within the A/B-domain [19, 20]. A further possible explanation could be the cross-reaction of C-terminal-targeted antibodies with ERß, because the C-terminal part of the molecule is considerably more highly conserved than the N-terminal A/B-domain between the two estrogen receptor types [21–23]. However, in a supplementary experiment no specific signals could be detected with the antibodies AER311 and HT277 in bovine granulosa cells, which are known to express ERß [24], whereas in ovarian stroma, positive reactions were readily detectable with C- and N-terminal targeted antibodies. Furthermore, the expression of an ER variant in the caruncular epithelium could also be considered; such a variant was found in the rat decidua [25]. However, only specific bands corresponding to full-size ER were found in a placentome in Western blot analysis using C- and N-terminally directed pmAb. Thus, the reason for the diverging staining patterns in immunohistochemistry remains unclear. However, taken together, the immunohistochemical results, the detection of ER-specific mRNA by RT-PCR, and Western blot analysis clearly suggest the expression of ER in the bovine caruncle.
According to the immunohistochemical results, the expression of ER in bovine placentomes is restricted to their maternal part. This is in contrast to an immunohistochemical study [26] published during submission of this paper, in which positive nuclear reactions were also detected in the tunica media of fetal blood vessels. In our study and using the same pmAb (HT277) that was used by Boos et al. [26], only occasionally was a diffuse, weak to moderate cytoplasmic signal observed in trophoblast cells (visible in Fig. 1B), which was not present in the respective negative control. However, because this cytoplasmic staining seen in trophoblast cells was not accompanied by nuclear signals, it was regarded as unspecific. Identification of ER in caruncular stromal and epithelial cells suggests that they are target cells of estrogens produced in the trophoblast and points to a role of placental estrogens as paracrine regulators of caruncular growth and differentiation. This hypothesis is supported by the fact that proliferative activity of the ER-expressing caruncular stromal cells investigated in a parallel study using identical samples [10] follows local estrogen tissue concentrations [27] in the second half of gestation. In addition, the extremely high proliferative activity [10] of the ER-positive caruncular epithelial cells, which are in direct contact with the estrogen-producing trophoblast cells suggests that placental estrogens may be involved in the stimulation of proliferation in this cell type.
In contrast to the study by Boos et al. [26], no positive reactions were observed in the tunica media of maternal vascular blood vessel walls. However, the occurrence of positive signals in sickle-shaped stromal cells directly attached to fine capillaries suggests that estrogens may also be indirectly involved in the regulation of caruncular angiogenesis and blood flow.
It is interesting that ER signaling in caruncular epithelium does not lead to a detectable up-regulation of progesterone receptor (PR) in these cells, whereas in the underlying ER-positive caruncular stroma, PR was clearly detectable in a preceding study [9] performed on identical samples.
The role of placental estrogens in the etiology of placental retention has been a controversial issue for decades [28]. In our study, a significant decrease in the proportion of ER-positive caruncular stroma cells and a decline in their staining intensity was found between Day 270 and parturition, with the exception of the area close to the caruncular stalk. Also, the number of ER-positive caruncular epithelial cells was considerably reduced due to the immediate prepartal dismantling of the caruncular epithelium [29]. This suggests that the bovine placentome is relatively unresponsive to estrogens at parturition, which is in accordance with earlier results obtained by ligand binding studies [30]. Thus, high placental estrogen levels at parturition are obviously indicative of a mature placenta with no or only marginal functions in respect to placental release.
In conclusion, the bovine placentomes express ER in caruncular stromal and epithelial cells, which are thus identified as putative targets of placental estrogens. Also taking into account features of proliferative activity in these cells [10], this suggests a role for placental estrogens as a paracrine factor involved in the regulation of placental growth and differentiation.
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ACKNOWLEDGMENTS |
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-overexpressing and sham-transfected HEK-293 cells. |
FOOTNOTES |
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1 Supported by the German Research Foundation (DFG) grant SCHU 1195/1–1 and the Ewald und Hilde Berge-Stiftung. 
2 Correspondence: Gerhard Schuler, Klinik für Geburtshilfe, Gynäkologie und Andrologie der Groß- und Kleintiere mit Tierärztlicher Ambulanz, Justus-Liebig-Universität Giessen, Frankfurter Strasse 106, D-35392 Giessen, Germany. FAX: 049 641 29328; gerhard.schuler{at}vetmed.uni-giessen.de
3 Current address: Solvay Pharmaceuticals Research Laboratories, Hans-Böckler-Allee 20, D-30173 Hannover, Germany
Accepted: November 1, 2001.
Received: March 2, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in target tissues. Eur J Cell Biol 2000; 79:383-393[CrossRef][Medline]This article has been cited by other articles:
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  G Schuler, G R Ozalp, B Hoffmann, N Harada, P Browne, and A J Conley Reciprocal expression of 17{alpha}-hydroxylase-C17,20-lyase and aromatase cytochrome P450 during bovine trophoblast differentiation: a two-cell system drives placental oestrogen synthesis. Reproduction, April 1, 2006; 131(4): 669 - 679. [Abstract] [Full Text] [PDF]
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