17β-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes

C2C12 cell culture

C2C12 murine myoblasts (MBs) (European Collection of Cell Cultures, ECACC No. 91031101, Salisbury, UK) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% heat-inactivated foetal bovine serum, 2 mM glutamine (Sigma) and 1% antibiotic antimycotic solution (Sigma). The cultured cells were maintained in 5% CO₂ atmosphere at 37 °C. To obtain differentiated myotubes (MTs), cells at 80% confluence were switched into differentiation medium (DM) containing 10% heat-inactivated horse serum.

Phenol red-free medium was used throughout the experiments to avoid its weak estrogen-like activity.

BT474 cell culture

BT474 (American Type Culture Collection, ATCC^®, HTB-20, Manassas, VA, USA) human mammary gland carcinoma cells were grown in DMEM supplemented with 10% heat-inactivated foetal bovine serum, 2 mM glutamine (Sigma) and 1% antibiotic antimycotic solution (Sigma) as positive control for the enzyme immunoassay (EIA). BT474 cells were grown in 100 mm petri dishes until 80% confluence, were starved and treated with E2 (10 nM) for 48 h to induce OXT secretion as described by Cassoni et al. (2006).

Experimental design

In all experiments, the sera were stripped with charcoal and dextran as described in Sharma, Handa & Uht (2012) to remove hormones.

To assess the physiological OXT and OXTR expression level, C2C12 MBs and MTs were respectively collected at day 3 and day 7. Subsequently, trials of acute and chronic administration of E2 were conducted on MTs to study gene expression modifications.

For the acute treatment trial, MTs were grown to day 6 and then starved with media containing stripped horse serum for 24 h before the E2 treatment (Sigma). The MTs were incubated with E2 (10 nM) at day 7 and then harvested at 0.5, 1, 2, 3, and 4 h of incubation (adapted from Sharma, Handa & Uht (2012)) to determine the Oxt and Oxtr expression levels. The control cultures were administered vehicle (Fig. 1).

For the chronic treatment trials, cells were treated with E2 (10 nM) or OXT peptide (10 µM) (Sigma), or a combination of E2 with OXT peptide (E2-OXT) (10 nM and 10 µM, respectively) every 48 h during differentiation from MB to MT (Fig. 1). OXT peptide was dissolved in 10 nM citrate buffer to increase its stability at high temperature (Avanti et al., 2011). Day 7 was chosen to harvest the differentiated MTs and to conduct the transcriptomic analysis because spontaneous contraction of MTs was observed, which is commonly associated with the maturation of MTs in culture. The levels of the Oxt, Oxtr, Myf6, Myf5, Myod, Myog Myh, Fabp3, Fabp4 and Pparg mRNAs were determined. Each experiment was performed five times in duplicate.

Myotube fusion index

The fusion index is a frequently used index of muscle cell differentiation and provides a measure of the proportion of the total cell populace that has fused (Agley et al., 2012). The fusion rate of the MTs that were chronically treated with E2, OXT and E2-OXT was evaluated at day 7 and compared to the untreated cell culture. The cells were washed with PBS and fixed in 10% neutral buffered formalin for 10 min. The fixed MTs were rinsed twice with PBS and stained with haematoxilin and eosin (H&E). Five control and five treated wells from independent experiments were analysed. For each well, the nuclei within the MTs and the nuclei of the unfused cells were counted in 10 randomly selected fields (200×) with Image Pro^® plus (Version 3.0.1) software. A cell containing three or more nuclei was considered to be a MT (Ge, Yu & Jiang, 2012). For each slide, the fusion index percentage was calculated as (the number of fused nuclei/total nuclei)*100.

RNA extraction and relative quantification by qPCR

The total RNA from each cell culture sample was extracted using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. The RNA concentration was determined by UV-Visible spectrophotometry and the RNA integrity was verified by automated gel electrophoresis system (Experion Instrument; Bio-Rad, Hercules, CA, USA). The cDNAs were synthesized from 1 µg of the total RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, D), which included DNase reaction, according to the manufacturer’s protocol.

To determine the relative amounts of specific Oxt, Oxtr, MRFs, Myh, Fabp3, Fabp4, and Pparg transcripts, the cDNAs were subjected to qPCR using the IQ5 detection system (Bio-Rad) and the IQ SYBR Green Supermix (Bio-Rad). The peptidylprolyl isomerase A (Ppia) cDNA was used as housekeeping gene control. The primer sequences were designed using Primer 3 (vers. 0.4.0) (Untergrasser et al., 2012) or Primer-BLAST (Ye et al., 2012), except for those for the Myh (Feng et al., 2009) and Ppia (Nishimura et al., 2008) genes, which were based on the literature (Table 1).

The levels of gene expression were calculated using the relative quantification assay based on the comparative C_q method (ΔΔC_q method) (Bustin et al., 2009). The relative abundances of each transcript were recorded as 2^−ΔΔCq (fold increase) (Pfaffl, 2004).

Regarding the comparison of physiological Oxt and Oxtr expression in untreated MBs and MTs, the gene expression levels were described as mRNA arbitrary units (2^−ΔCq).

Western blot analysis

The total proteins were extracted from cell lysate using RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS and 2 mM EDTA) supplemented with a protease inhibitor cocktail (Sigma). The protein concentration was determined using the Bio-Rad DC Protein Assay. Proteins were boiled at 95 °C for 5 min in Leammli buffer and 35 µg of the total protein were resolved by 10% SDS–PAGE for OXTR assessment. The proteins were blotted to Hybond-P PVDF membrane (Amersham Biosciences, Piscataway, NJ, USA) using the Mini Trans-Blot cell (Bio-Rad). The blotted membranes were blocked with bløk-CH Buffer (Millipore KGaA, Darmstadt, D) for 1 h at room temperature, followed by overnight 4 °C incubation with the primary goat polyclonal anti-OXTR antibody (1:200; Santa Cruz Biotechnology). Proteins of rat heart was used as positive control (C+) for OXTR as other authors described (Jankowski et al., 1998). The membranes were subsequently incubated with a secondary horseradish peroxidase (HRP)-conjugated anti-goat antibody (1:10000), developed using the SuperSignal West Pico IgG Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA) and signal was recorded on CL-XPosure X-ray film (Thermo Fisher Scientific) or by Chemi-Doc MP System (Bio-Rad). α-tubulin (1:10000, clone B-5-1-2; Sigma) was used as total protein loading control. Densitometric analysis of the bands was performed by the public domain ImageJ software (US National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/nih-image/), (n = 3).

Enzyme immunoassay (EIA)

To study OXT peptide expression, the culture media from MBs and MTs, from the acutely treated MTs and BT474 were collected and immediately stored at −80 °C until further analysis. The soluble OXT peptide concentration was measured by enzyme immunoassay methodology using the EIA kit developed by Cayman Chemical (Tallinn, EE). The manufacturer reports a limit of detection (LOD) for this kit of approximately 18 pg/ ml. OXT was extracted from 10 ml of culture medium aliquots using a solid-phase extraction (SPE), 1,000 mg C-18 Sep- Pack column (Supelco; Sigma-Aldrich, St. Louis, MO, USA) as previously described (Divari et al., 2013). Then, concentrated samples were collected in a glass tube and evaporated to dryness under a stream of nitrogen gas; they were reconstituted in 0.5 ml of assay buffer and immediately measured (concentrated 20-fold). The absorbance at 405 nm was read using Microplate Reader 680 Model (Bio-Rad) and a standard curve was created using a four parameter logistic function.

Statistical analysis

All cell culture experiments were conducted in duplicate and were repeated five separate times.

All statistical analyses were performed using the GraphPad Prism 4 (vers. 4.03) software (GraphPad Inc., San Diego, CA, USA). Normal distribution was tested by the Kolmorov-Smirnov test. Grubbs’ test was used to determine and exclude potential outliers. In the qPCR experiments in which results were expressed as 2^−ΔCq, a paired t-test or Wilcoxon test were applied. For qPCR results expressed as differences in the relative expression (2^−ΔΔCq) of each target gene between the hormone-treated and untreated cells, data were analysed by ANOVA or Kruskal–Wallis for the acute trial, followed by Dunnett’s or Dunn’s post test, and by the paired t-test or Mann–Whitney test for chronic trial results. Data are presented as the means ± SEM from 5 experiments. p < 0.05 was considered significant.

Basal levels of OXT and OXTR expression during differentiation of C2C12 MBs into MTs

The basal levels of Oxt mRNA expression were extremely low in MBs (3.47E−06 mRNA arbitrary units); while, during myogenic differentiation into MTs, the Oxt mRNA concentration increased (5.01E−06 mRNA arbitrary units, p < 0.05). Similarly, Oxtr mRNA expression in MBs was scarse (8.40E−07 mRNA arbitrary units) and increased in differentiated MTs (4.28E−06 mRNA arbitrary units, p < 0.001) (Fig. 2A). Protein expression of OXT in MBs and MTs was demonstrated by EIA test (1.50 pg/ml in MBs and 4.26 pg/ml in MTs respectively) (Fig. 2C). OXTR protein was demonstrated and quantified by western blot (Figs. 2B and 2D).

Effect of acute E2 administration on OXT and OXTR expression in C2C12 MTs

In the acute assay, Oxt mRNA was upregulated by approximately 3-fold after 0.5 h of E2 incubation (p < 0.01) compared with the control cells. After 3 and 4 h of E2 treatment, the mRNA levels progressively decreased until control levels (Fig. 3A). Oxtr mRNA levels were not significantly regulated by treatment (Fig. 3B).

The EIA test on the growth media from the examined cell culture showed different OXT peptide concentration between samples: 7.61 pg/ml of OXT peptide were detected after 0.5 h of E2 administration against the 4.26 pg/ml of OXT of the control sample. The media from BT474 E2-treated cells was used as positive control for OXT secretion demonstrating a total OXT concentration of 6.90 pg/ml (Fig. 3C). OXTR protein expression was studied by Western blot (Figs. 3D and 3E) and no statistically significant differences were recorded.

Effect of chronic hormone administration on MTs fusion index

To study the effect of chronic E2, E2-OXT and OXT treatments on myogenic development fusion index was studied in MTs and representative images are shown in Figs. 4A and 4B. The fusion index was not affected by chronic administration of E2 nor by OXT or the combination of both molecules (Fig. 4C).

Effect of chronic hormone administration on Oxt, Oxtr, MRFs, Myh, Fabp3, Fabp4 and Pparg gene expression in C2C12 MTs

The MRF mRNA levels in the C2C12 MTs were not significantly regulated by any of the chronic treatments (Figs. 5A–5C). A mild downregulation of the Myh gene was observed in the chronically treated cells; in particular E2 induced a significant decrease of approximately 0.90-fold compared with the control culture (p < 0.05) (Fig. 5A). Chronic treatments with E2-OXT and OXT did not significantly influence the expression of the studied genes involved in growth and differentiation of the MTs.

Chronic administration of E2 and E2-OXT induced a significant overexpression of the Oxtr gene. In particular, in the E2-treated cells the levels of the Oxtr mRNA were increased by approximately 29-fold compared with the control culture (p < 0.05) (Fig. 5D) and the combination of the two hormones induced this mRNA by approximately 11-fold compared with the control culture (p < 0.05) (Fig. 5F).

Moreover, chronic administration of E2-OXT in MTs induced a significant overexpression of Fabp4 (2.66-fold) (Fig. 5I), which is involved in fatty acid handling, while a decrease in the expression of Fabp4 (0.62-fold) was observed in the OXT-treated MTs (Fig. 5H)