Estrogen receptors alpha and beta in rat placenta: detection by RT-PCR, real time PCR and Western blotting

The polyclonal antibody corresponding to amino acids 1–150 of the ERβ was purchased from Santa Cruz Biotechnology, Inc., U.S.A (catalogue # H-150; sc-8974). Two monoclonal antibodies raised against different epitopes of the ERα were used: one raised against the steroid binding domain of the ERα (amino acid residues 495–595; referred to as ERα-S; catalogue # C-311; Santa Cruz Biotechnology, Inc., U.S.A) and the other raised against the hinge region (amino acid residues 287–300; referred to as ERα-H; catalogue # SRA-1000; Stressgen Biotechnologies Corp., Canada). The actin monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc., U.S.A (catalogue # C-2). PVDF membranes were obtained from Amersham Pharmacia Biotech Ltd., U.K.

Sprague-Dawley rats were obtained from Bantin and Kingman (U.K.). The rats were housed in the Animal Resources Centre at the Faculty of Medicine, Kuwait University and had free access to food and water. The rats were maintained on a cycle of 12 h light and 12 h darkness at 22°C. The experiments were carried out in accordance with the rules of laboratory animal care in this institution.

Female rats were mated with males and mating was verified by the presence of sperm in the vaginal smear; this was designated as day 0 of pregnancy. Pregnant dams were stunned and killed by cervical dislocation at 16, 19 or 21 days gestation (dg). Uterine horns containing conceptuses were removed and placed immediately on ice. Fetuses and placentae were separated and placental tissues from each litter were pooled (four pregnancies were obtained at each gestational age [n = 4] and three to four placental tissues per pregnancy were pooled). The maternal uterus and maternal brain of the 21 dg dams were used as a positive control (data not shown). The cryoprotective agent, dimethylsulfoxide, (10% v/v) was added before freezing at -70°C for subsequent analysis.

Samples were homogenized in denaturing solution (4 M guanidine thiocyanate salt, 25 mM sodium citrate, pH 7.0, 0.5% w/v sarcosyl and 0.1 M 2-mercaptoethanol) using a sterile hand-held homogenizer. To 3.6 ml homogenate, 0.36 ml sodium acetate (2 M, pH 4.0), 3.6 ml citrate buffer-saturated phenol (pH 4.3) and 0.72 ml chloroform:isoamyl alcohol (49:1) were added sequentially, shaking well between each addition. Tubes were vigorously shaken for 15 s after the final addition. Tubes were kept on ice for 15 min, then centrifuged (10,000 g for 20 min at 4°C). The aqueous phase was removed, avoiding the DNA interphase, and an equal volume of ice-cold isopropanol was added. After shaking, tubes were kept at -20°C for > 1 h. The precipitate was collected by centrifugation (10,000 g for 20 min at 4°C) and dissolved in 0.3 ml denaturing solution. Nucleic acid was re-precipitated by adding an equal volume of ice cold-isopropanol. After > 1 h at -20°C, the samples were centrifuged (10,000 g for 10 min at 4°C). The pellet was washed twice with 1 ml 75% (v/v) ethanol (at -20°C) by suspension/centrifugation. The final pellet was air-dried, then dissolved in 0.5% (w/v) SDS (0.25 μl/mg wet weight tissue) at 65°C for 15 min. Extracted RNA was stored at -70°C. The quality and quantity of total RNA sample was determined using spectroscopic measurements at 260 and 280 nm. Samples with A₂₆₀/A₂₈₀ ratios > 1.7 were only studied further. The integrity of total RNA was checked by agarose gel electrophoresis and 28S and 18S rRNAs visualized after ethidium bromide staining.

SDS was removed from total RNA by precipitation with sodium acetate-isopropanol then resuspended (at ca. 1 μg/μl) in water. The RNA concentration was determined by spectrophotometery and was adjusted to 0.5 μg/μl with water. All samples were DNase treated before reverse transcription. Briefly, 2 μg of total RNA was mixed on ice with 40 U of RNasin, 1 U of DNase and 1× DNase buffer in a final volume of 20 μl. The mixture was left at room temperature for 15 min and the reaction was terminated by adding 2 μl of 25 mM EDTA and heating at 70°C for 10 min. The DNase-treated sample was divided into two 11 μl aliquots, 100 ng random hexamers were added and the mixture was heated at 70°C for 10 min, immediately chilled on ice for > 3 min then briefly centrifuged. With the tube on ice, the following were added: 1× first strand buffer (25 mM Tris-HCl pH 8.3, containing 37.5 mM KCl and 1.5 mM MgCl₂), 5 mM DTT and 500 μM dNTP mix. To one tube (RT+ reaction) 200 U Superscript II RNase H^- reverse transcriptase were added, whereas water was added to the other tube (control RT-reaction) in a final volume of 20 μl. After gentle mixing, reactions were incubated at room temperature for 10 min then at 42°C for 50 min. Reactions were terminated by heating at 70°C for 15 min.

The PCR reaction was carried out in a programmable thermal cycler (Perkin Elmer, model 9700). The ERα primer sets used were rERα U (5'-TAAGAACCGGAGGAAGAGTTG) and rERα L (5'-TCATGCGGAATCGACTTG) that give an expected 623 bp product. The 18S housekeeping gene primer sets r18S U (5'-GTCCCCCAACTTCTTAGAG) and r18S L (5'-CACCTACGGAAACCTTGTTAC) give an expected 419 bp product. The reaction mixture consisted of: 1× PCR buffer (20 mM Tris/50 mM KCl), 3 mM MgCl₂, 0.5 mM dNTPs and 0.3 μM each of upper and lower primers, 0.5 μl template (RT⁺, or RT- in addition to a water sample used as a negative control) and 1.25 U recombinant Taq DNA polymerase in a final volume of 25 μl. The PCR reactions were then cycled as follows: 5 min at 94°C (1 cycle); 30 s at 94°C (denaturation step), 30 s at the 45°C (annealing step) and 1 min at 72°C (extension step) for the required number of cycles (24 cycles for 18S and 32 cycles for ERα). Tubes were then incubated for a further 7 min at 72°C (1 cycle).

The ReT-PCR reaction was carried out in a ReT-PCR system (Applied Biosystems, model 7500). The endogenous control, β-glucuronidase, used in this experiment was supplied by Applied Biosystems. The ERβ primer sets used detect both the wild type and a variant that has a 54 bp (18aa) in-frame sequence between exons 5 and 6 of wild type ERβ [18]. These primer sets were rERβ LBD U (5'-GAGCTCAGCCTGTTGGACC) and rERβ LBD L (5'-GGCCTTCACACAGAGATACTCC) [19]. The PCR reactions were prepared using the Taq Man universal master mix (# 4324018, Applied Biosystems) then cycled as follows: 2 min at 50°C (1 cycle); 10 min at 95°C (1 cycle), 15 s at 95°C and 1 min at 60°C for 60 cycles.

Placentae were thawed and washed twice with ice-cold isotonic saline then homogenized in ice-cold homogenization buffer containing glycerol [to stabilize the receptors] and protease inhibitors (10 mM Tris-HCl pH 7.4, 1.5 mM EDTA and 10% v/v glycerol, 1.0 mM DTT, 1 μg/ml leupeptin, 100 μg/ml bacitracin, 2 μg/ml aprotonin, 1 μg/ml pepstatin A), using a Polytron homogenizer, to yield a 5% (w/v) homogenate (the total receptor fraction). Homogenates were passed through a nylon mesh (110 μm pore size) then through steel gauze (40 μm pore size). An aliquot was removed for protein assay. Samples (50–100 μg protein) and molecular weight rainbow markers (14,300 – 220,000 daltons; Amersham Pharmacia Biotech Ltd., U.K.) were mixed with loading buffer (0.03% w/v bromophenol blue, 32 mM Tris-HCl pH 6.8, 8% w/v dithiothreitol and 4% w/v SDS), boiled for 5 min, then chilled on ice and centrifuged (10,000 × g for 1 min). Samples were then electrophoresed (SDS PAGE; 7.5% polyacrylamide gels). The distance migrated by the marker proteins and bromophenol blue front were measured. The buffer chamber of the electrophoresis apparatus was filled with degassed transfer buffer (25 mM Tris-HCl, 150 mM glycine, 20% v/v methanol and 0.1% w/v SDS, pH 8.3). Gels were equilibrated in transfer buffer then protein was transferred to PVDF membranes with a constant current of 300 mA overnight with cooling. After transfer the membranes were dried and stored at 4°C.

Membranes were blocked for 1 h at room temperature with 10% non-fat dry milk in TBS-T (20 mM Tris, 137 mM NaCl, pH 7.6 and 0.1% v/v Tween). The membranes were washed by rinsing twice with TBS-T, followed by two 10 min washes (20 ml/wash). Membranes were then incubated overnight at 4°C with 3 ml primary antibody solution diluted in 5% w/v non-fat dry milk in TBS-T (ERα-S diluted 1:500, ERα-H diluted 1:1000 and ERβ antibody diluted 1:1000). After incubation, the membranes were rinsed twice with TBS-T followed by one 15 min wash and two 5 min washes (20 ml/wash). Membranes were incubated with the appropriate secondary antibody (anti-mouse Ig HRP linked diluted 1:20000 or anti-rabbit Ig HRP linked diluted 1:10000 in TBS-T) for 1.5 h at room temperature then rinsed twice with TBS-T followed by one 15 min wash and four 5 min washes (20 ml/wash). Detection was by chemiluminescence after incubation with an HRP-linked secondary antibody – using a commercial kit (ECL-Plus; Amersham Pharmacia Biotech Ltd., U.K.) and standard methodology. After obtaining the results for the respective estrogen receptor primary antibodies, the membranes were stripped in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) at 50°C for 30 min with occasional shaking. The membranes were then washed twice in TBS-T for 10 minutes each before being re-probed with anti-actin antibody (diluted 1:500 in TBS-T) which served as an internal control. All results were expressed relative to actin.

Protein was determined by a dye-binding method using Coomassie Plus Protein assay reagent (Pierce & Wariner Ltd, U.K.)

In order to determine the sizes of the bands a marker lane was always included. The distance migrated by the protein marker was measured and a standard curve constructed using a plot of log₁₀ molecular mass versus relative mobility (distance migrated by band ÷ distance migrated by dye; R_f). The size of the unknown protein band was then determined using the equation of the line.

After the RT-PCR reaction for ERα, PCR products were electrophoresed alongside a 100 bp DNA marker (100 bp ladder, Gibco), through a 2% (w/v) low electroendosmosis (LE; Boehinger Manheim) agarose gel stained with ethidium bromide. All images were captured using Gene Genius Bio Imaging System and od values of PCR products were measured using Gene Tools Software. As for ReT-PCR the results for the average CT threshold cycle for each sample was used to express the relative expression of ERβ to β-glucuronidase.

Statistical analysis was performed using SPSS (ANOVA followed by post-hoc analysis [LSD]) when the test for homogeneity of variance was fulfilled and using Games-Howell post-hoc analysis when the homogeneity of variances was not attained. All data shown are mean ± S.E.M. and a p value of < 0.05 was taken as the minimum level of significance.

As expected there was a significant increase (p < 0.001) in placental weights and fetal body weights with gestation (Table 1). As for the litter number, there was a significant decrease (p < 0.001) between 16 and 21 dg and fetal resorptions were detected in some dams, however, these dams were not included in this study.

There was no significant change in placental protein concentration (Table 1), nevertheless, a significant increase in protein content was observed between 16 and 19 dg (p < 0.001). This increase was in parallel with the increase in placentae weight noticed with gestation (Table 1).

The expected PCR product (623 bp) for the ERα mRNA was detected in rat placentae from as early as 16 dg (Figure 1). However, a ~500 bp product was also detected from 16 dg. The RT- and W reactions did not show any bands (figures not shown), indicating that the amplified products in RT+ reactions are not due to genomic DNA nor due to contamination of reagents, respectively. The od measurements of PCR products obtained for ERα gene were expressed relative to the od obtained from 18S. When expressed relative to the 18S housekeeping gene it was seen that the pattern of expression of both products relative to 18S was similar with a significant decrease in ERα mRNA expression between 16 and 21 dg and 19 and 21 dg (*p < 0.001).

Using RT-PCR it was not possible to detect ERβ mRNA expression. However, using ReT-PCR it was possible to amplify the ERβ gene (Figure 2). The average threshold cycle (C_T) for duplicate samples was expressed relative to that of β-glucuronidase (endogenous or housekeeping gene). No significant change in ERβ gene expression was detected with gestation.

A ~67 kDa single band for ERα was observed for the placental samples using both the ERα-H and the ERα-S antibodies from as early as 16 dg (Figures 3 &4, respectively). However, using the ERα-H antibody, another band of apparent molecular weight of 54 kDa was detected. The abundance of these ERα variants decreased significantly between 16 and 19 dg (p < 0.05) as shown when the ERα-H antibody was used. However, using the second antibody, ERα-S, no apparent change in protein expression was observed although the level at 19 dg appeared lower but did not reach significance. The 54 kDa band observed with the ERα-H antibody was more abundant at 16 dg compared to the 67 kDa (Figure 3). This difference in expression was normalized by 19 dg.

When studying the expression of the second estrogen receptor isoform, ERβ, four bands were detected with apparent molecular weights of 76, 59, 54 and 41 kDa (Figure 5). When comparing the level of expression of the ERβ protein as pregnancy progressed, it was found that there was a significant decrease in expression of the 59 and 54 kDa bands (p < 0.001) between 16 and 19 dg. Moreover, the 76 kDa band showed a tendency to decrease at 19 dg, however, this was not statistically significant. On the other hand, the 41 kDa ERβ variant showed a different pattern of expression with a trend to increase between 16 and 19 dg (not statistically significant).