Reagents and chemicals
The 17b-estradiol (E2), dexamethasone (DEX), progesterone (P4), mifepristone (RU486), and corn oil were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA), and the ICI 182,780 was obtained from Tocris (Avonmouth, UK). Stock solutions were diluted with dimethyl sulfoxide (DMSO; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Animal treatment
Specific pathogen-free immature female Sprague-Dawley rats weighing 15–20 g at postnatal day 10 were purchased from Samtako (Osan, Republic of Korea). The rats were housed in polycarbonate cages and were allowed to acclimate to their housing in an environmentally controlled room (temperature 23°C ± 2°C, relative humidity 50% ± 10%, frequent ventilation, and a 12 h light:dark cycle).
After approximately 1 week of acclimatization the rats were separated into seven groups (n = 7) and each group was subcutaneously (SC) injection with E2 (50 µg/kg body weight [BW]), DEX (10 mg/kg BW), P4 (20 mg/kg), or vehicle (corn oil) once per day for 5 consecutive days. To evaluate the affected signal pathways, antagonist groups were injected with ICI 182,780 (10 mg/kg BW) and RU 486 (50 mg/kg BW) 30 min prior to hormone administration. At 12 h after final injection, all immature rats were euthanized with ether and tissue samples were collected. All experimental animal management and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University.
RNA extraction and real-time PCR
Rats were euthanized by CO2 asphyxiation as described in previously [19]. The brains were removed and sagittally divided and the olfactory bulbs, cerebral cortex, hypothalamus, cerebellum, and brain stem were dissected from the right hemisphere. Total RNA was extracted using the TRIzol (Ambion, Austine, TX, USA), according to the manufacturer’s instructions. RNA purity was determined using an Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA). Next, first strand cDNA was prepared by subjecting 1 µg total RNA to reverse transcription using Moloney murine leukemia virus reverse transcriptase (ThermoFisher Scientific, Logen, UT, USA) with random primers (9-mers; TaKaRa Bio Inc., Kusatsu Japen). The cDNA template (2 µL) was added to 10 µL of 2× SYBR premix Ex Taq (TaKaRa Bio) and 10 pmol of each specific primer for ERa, PR, and GR genes. The primer sequences are showed in additional table 1. By using an ABI 7300 Real-time PCR system (Applied Biosystems, Foster City, CA, USA), quantitative real-time PCR was conducted under the following conditions: 40 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s. Fluorescence intensity was measured at the end of the extension phase of each reaction cycle, and the reaction cycle at which the PCR products exceeded the fluorescence intensity threshold in the exponential phase of PCR amplification was considered the threshold cycle (CT). Each data point was analyzed to the internal control (GAPDH) gene for determination of a normalized arbitrary value. Relative expression (R) was calculated by using the equation R = 2-ΔΔCT and was normalized to the vehicle.
Immunofluorescence assay
Immunofluorescence assay was performed as described previously [20, 21]. For examination of TRPV5, TRPV6, NCX1, and PMCA1 immunofluorescence, rat brain was fixed with 4% paraformaldehyde and serial 80 μm think coronal and sagittal sections were cut on a vibratome (Leica, VT1000S, Nussloch, Germany). To improve antibody penetration, brain sections were soaked in 0.5% Triton X-100 for 15min and incubated in blocking solution (5% normal goat serum, normal donkey serum and 0.3% Triton X-100) for 1 h. Primary antibodies used were rabbit anti-TRPV5 antibody (Allomone labs, Israel, ACC-035, 1:500), rabbit anti-TRPV6 (Allomone labs, A2052, 1:500), mouse anti-NCX1 (Swant, Bellinzona, Switzerland, R3F1, 1:500), rabbit anti-PMCA1 (Swant, PMCA1, 1:500), rabbit anti-ERα (Santa Cruz Biotechnology, CA, USA, SC-542, 1:500), mouse anti-ERα (ThermoFisher Scientific, Waltham, MA, USA, MA5-13304, 1:500), rabbit anti-PR (Santa Cruz, SC-538, 1:500), mouse anti-PR (Abcam, Cambridge, UK, ab2765, 1:500), rabbit anti-GR (Santa Cruz, SC-1004, 1:500) and mouse anti-GR (Abcam, ab2768, 1:500) for 24 h at 4°C. Following washing in PBS, appropriate secondary antibodies conjugated with Alexa Fluor dyes (Invitrogen, Carlsbad, CA, USA, 1:1000) were used to detect primary antibodies, and DAPI (ThermoFisher, D1306, 1:1000) was used to stain nuclei.
Western blot assay
Brain proteins were extracted with Pro-prep (iNtRON Bio, Sungnam, Kyeonggido, Republic of Korea), according to the manufacturer’s protocol. Protein concentration was determined by using the BCA assay (Sigma-Aldrich). Next, a 30-40 µg sample was resolved by performing tris-glycine SDS-polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Taunton, MA, USA). Next, the membrane was blocked for 1 h with 5% skim milk dissolved in TBS-T. The membrane was incubated in primary antibodies diluted in 1% BSA for overnight at 4°C. Primary antibodies to rabbit anti-TRPV5 (Allomone labs, 1:1000), rabbit anti-TRPV6 (Allomone labs, 1:1000), mouse anti-NCX1 (Swant, 1:1000), rabbit anti-PMCA1 (Swant, 1:1000) and GAPDH (Santa Cruz, Biotechnology, 1:1000) were used. The membrane was washed four times for 10 min each with TBS-T. Horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling, Beverly, MA, USA, 1:3000) and anti-mouse IgG (Cell Signaling, 1:3000) were used as a secondary antibody with 2.5% skim milk dissolved in TBS-T for 60 min. The membrane was washed with TBS-T four times as previously mentioned, and immunoreactive proteins were visualized by using the West-One Western Blot Detection System (iNtRON Bio), according to the manufacturer’s instructions. Signals were detected with the Chemi Doc EQ (Bio-Rad, Hercules, CA, USA) imaging system and were analyzed by the Quantity One program (Image J, version 1.48; Wayne Rasband, NIH, Bethesda, MD, USA).
Data analysis
Data were analyzed by applying a nonparametric one-way ANOVA followed by Tukey’s correction test for multiple comparisons. All experiments consisted of three separate trials and each used a minimum of four samples. Data were analyzed using GraphPad Prism (v.5.0; GraphPad Software, La Jolla, CA) and are presented as mean ± S.E.M values.