Prostaglandin receptors EP and FP are regulated by estradiol and progesterone in the uterus of ovariectomized rats

Estradiol-17β and progesterone were purchased from Sigma Chemical Co. St. Louis, MO, USA. The hormones were dissolved in 99.5% ethanol at a high concentration and then diluted with propylene glycol to the required concentration. The final concentration of ethanol in the injections was less than 5%. The ERα selective ligand 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and the ERβ selective ligand 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) were purchased from Tocris Bioscience, Bristol, UK. RNAlater^® was purchased from Ambion, Austin, TX, USA.

Adult female rats (55-60 days old, approx. 250 g) of the Sprague-Dawley strain (BK-Universal, Sollentuna, Sweden) were used in all four experiments. The animals were housed in a controlled environment at 20°C on an illumination schedule of 12L: 12D. The rats had unlimited access to standard pellet food and water. Rats were ovariectomized and given two weeks to rest before proceeding with the treatments. The studies were approved by the committee on animal care in Sweden.

The ovariectomized rats (n = 22) were divided into four groups. Three groups were treated with E2 subcutaneously at doses 1.0 μg (n = 4), 2.5 μg (n = 6) or 5 μg (n = 6) per rat and day for two days. The control group (n = 6) was treated with vehicle alone.

The ovariectomized rats (n = 27) were divided into four groups and treated subcutaneously with 2.5 μg of E2 daily for 1 day (n = 7), 4 days (n = 7) or 7 days (n = 6). The control group (n = 7) received vehicle alone.

Forty-two ovariectomized rats were divided into seven groups (n = 6). They were treated by subcutaneous injections with either vehicle, 2.5 μg of E2 per rat and day, and/or 1 mg P4 per rat and day. They were treated with E2 and/or P4 for 24 or 48 hours. The treatment sequence is shown in Table 1.

Four groups of ovariectomized rats (n = 31) were treated with vehicle, E2, ERα selective or ERβ selective agonists, PPT or DPN, respectively. The animals were subcutaneously administered either 5.0 μg of E2 (n = 8) or 1.25 mg of PPT (n = 8) or 3.125 mg of DPN (n = 7) or vehicle (n = 8). The rats were killed 24 h after treatment. The doses of PPT and DPN were chosen by comparing data on the activity of the agonists and were prepared as reported earlier [14].

At sacrifice, the rat uterus was removed, stripped of fat and connective tissue. In study 1 and 4 one portion was stored in RNAlater^® for RNA preparation and another portion was immersion fixed in buffered formalin at room temperature (RT) overnight and then stored in 70% ethanol in 4°C to be embedded for immunohistochemistry. In study 2 and 3, tissues were only prepared for immunohistochemistry.

Total RNA from uterine tissues were purified with RNeasy^® Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol for fibrous tissues. The protocol included an on-column DNase digestion, as recommended by the manufacturer. Two micrograms of total RNA from each sample was reverse transcribed at 37°C for 60 min in a final volume of 20 μl with a reaction mixture (Qiagen) containing 1 × RT buffer, dNTP mix (0.5 mM each dNTP), 600 ng random primers (Invitrogen, Paisley, UK), 30 units RNase inhibitor (Qiagen GmbH, Hilden, Germany), and 4 U of Omniscript™ reverse transcriptase (Qiagen).

The oligonucleotide primers for EP1, EP2, EP3, EP4, FP and Rplp0 (housekeeping gene) are presented in Table 2, along with their predicted sizes. Real time PCR was performed in an iCycler™ iQ Real Time PCR System (Bio-Rad Laboratories, Inc, CA USA). For PCR, the cDNAs corresponding to 50 ng RNA were added to 12.5 μl of iQ™ SYBR^® Green Supermix (Bio-Rad) and 0.3 μM of each oligonucleotide primer in a final volume of 25 μl. After initial incubation for 3 min at 95°C, the samples were subjected to 40 cycles of 10 s at 95°C, followed by 45 seconds annealing at 57 to 64°C depending upon the genes (Table 2). All PCR assays were performed in duplicates. The purity of PCR products was confirmed by a melting curve analysis in all experiments. Each PCR assay included a negative control containing an RNA sample without reverse transcription. The primer pairs (Table 2) were designed with NCBI/Primer-BLAST program.

Paraffin embedded blocks were cut in 5 μm thick sections and mounted on histological slides. The paraffin sections were deparaffinized, rehydrated and washed with phosphate-buffered saline (PBS; pH 7.4). Antigen retrieval was performed by boiling the sections in 0.01 M sodium citrate buffer (pH 6.0) in microwave for 10 min. The endogenous peroxidase activity was quenched by placing the slides in 3% hydrogen peroxide in methanol. The slides were then blocked with 1.5% normal donkey serum followed by incubation with primary antibodies. Primary antibodies (Cayman Chemicals, Michigan, USA) were incubated overnight at 4°C. The antibodies were incubated by diluting to final concentrations of 3 μg/ml for EP1 (Cat # 101740), 2 μg/ml for EP2 (Cat # 101750), 1.5 μg/ml for EP3 (Cat # 101760), 10 μg/ml for EP4 (Cat # 101775) and 5 μg/ml for FP (Cat # 101802). The specificities of the antibodies were verified by performing western blot using rat uterine extracts (data not shown). These antibodies have been used and verified before in rat tissues [15, 16]. The negative controls were obtained by replacing the primary antibody with an equal amount of rabbit IgG. Thereafter, incubation with biotin conjugated donkey anti-rabbit secondary antibody (SantaCruz, CA, USA) was performed at RT for 30 min at a dilution of 1:1000. Further, incubation with avidin-biotin horseradish peroxidase complex was performed at RT for 30 min using Vectastain elite^® ABC kit (Vector labs, CA USA). The location where the enzyme was bound was visualized by the addition of the substrate 3,3'-diaminobenzidine (DAKO, CA, USA), a chromogen which produces a brown insoluble precipitate. The sections were then counterstained with haematoxylin, which was followed by dehydration and mounting with Pertex. A Leica microscope was used to evaluate the slides. The staining intensities were assessed semi-quantitatively by manual scoring. All the slides were evaluated for the five different tissue compartments (luminal epithelia, glandular epithelia, stroma, myometrium and blood vessels). Scoring was performed independently by two investigators blinded to the identity of the slides. The staining intensity was arbitrarily graded on a scale of (0) no staining, (1) faint, (2) moderate and (3) strong.

Statistical analyses for all studies were carried out by Kruskal-Wallis test (ANOVA on ranks) and the significances were evaluated by Dunn's test using Sigma plot^® software. Values are considered significant when P < 0.05.

Real-time PCR data showed that E2 downregulates the expression of EPs and FP mRNAs in the uterus (Figure 1). The decrease was significant (p < 0.05) for EP1 and EP4 at low dose treatment (1.0 μg); EP2, EP3 and FP at mid dose (2.5 μg) and for the expression of all receptors when treated with the highest dose (5.0 μg). However, results from immunohistochemistry showed that there is an increase in the immunostaining of EP2 and EP3 following E2 treatments (Figure 2 and 3). There is an increase (p < 0.05) in the immunostaining of EP2 when treated with 5.0 μg of E2 in luminal epithelium, glandular epithelium, stroma and myometrium when compared to controls. In blood vessels a similar tendency was seen. EP3 immunostaining was increased (p < 0.05) in stroma and myometrium when treated with 2.5 μg of E2 (Figure 3). Corresponding tendencies could be observed in stroma and myometrium of study 3 after a comparable E2 treatment. Luminal epithelium, glandular epithelium and blood vessels also show similar trends. Immunostaining of the EP1, EP4 and FP receptors did not show any significant differences (data not shown).

Immunohistochemical scores showed a time depended upregulation of EP2 in rat uteri when treated with 2.5 μg of E2 (Study 2). An increase (p < 0.05) in immunostaining could be observed in myometrium from day 1 but in luminal epithelium, glandular epithelium and stroma the increase was significant after day 4 (Figure 4). The increase (p < 0.05) was significant in all the cell types including the blood vessels after 7 days of treatment with E2. Our observations from studies 1 and 3 show that EP2 is not upregulated after treatment with 2.5 μg of E2 for 2 days (Figure 3) or 1 day respectively, except for myometrium on day 1 (Figure 5). The myometrial EP2 expression showed similar upregulation after day 1 in both study 2 (Figure 4) and study 3 (Figure 5) where a similar dose of E2 was used. The other PG receptors examined did not show significant regulation after E2 treatment (data not shown).

In general, it was observed that there is a strong tendency for increased immunostaining of EP1 in all the compartments when treated together with E2 and P4, irrespective of the sequence of the treatments (Figure 5). In stroma we could observe an increase in immunostaining (p < 0.05) when treated with E2 followed by P4, when compared to vehicle treated control or E2 alone. Further, in myometrium an increase in immunostaining (p < 0.05) could be observed between the controls and groups treated with E2 and P4 irrespective of the treatment sequence. EP3 expression was upregulated (p < 0.05) when treated with P4 after priming with E2 in glandular epithelium, stroma and myometrium (Figure 5). Luminal epithelium and blood vessels showed similar tendencies although not statistically significant (data not shown). EP2 was upregulated (p < 0.05) in myometrium when treated with E2 alone after 24 h (Figure 5) but there was no change in the other cell types in any of the combined treatments. Treatment with P4 alone did not affect the regulation of any of the PG receptors investigated in this study. EP4 and FP did not show any regulation when treated with E2 or P4 alone or in combination.

Our results from real-time PCR analyses show that 5.0 μg of E2 and the ERα agonist PPT downregulated (p < 0.05) mRNA expression for all EPs and FP (Figure 6). Treatment with the ERβ agonist DPN resulted in a downregulation (p < 0.05) of EP2 and EP4 mRNA expressions (Figure 6). However, immunohistochemical scores showed an upregulation (p < 0.05) of the EP2 and EP4 proteins in stroma when treated with E2 (data not shown).

A summary of immunohistochemical results of the significantly regulated receptors from all the four studies are given in table 3.