The modulatory effect of visfatin (NAMPT) on the transcriptomic profile of porcine anterior pituitary cells during the peri-implantation period
The modulatory effect of visfatin (NAMPT) on the transcriptomic profile of porcine anterior pituitary cells during the peri-implantation period
For the experiment, the glandular parts of the pituitaries were obtained from gilts (age of 7-8 months and weight of 130-150 kg) on days 15 to 16 of gestation, and descended for slaughter at the commercial abattoir. Mating occurred on days 1 to 2 of the oestrous cycle, with the day following mating marked as the first day of pregnancy. The stage of gestation was confirmed by the presence, size, and morphology of conceptuses obtained through flushing from the uterine horns (L. L. Anderson, 1978).   The cells were washed with Dulbecco's medium (Sigma-Aldrich, USA) enriched with 0.1% bovine serum albumin (Sigma-Aldrich, USA) and antibiotics (Antibiotic Antimycotic Solution, Merck, USA), and homogenized with scalpel blade. The homogenates were digested with 0.2% collagenase solution (Sigma-Aldrich, USA) at 37°C for 30 min. Isolated APc were centrifuged three times at (1000 × g, 10 min, room temperature) in 15 mL tubes containing Dulbecco's medium. The remained tissue was treated with 0.2% collagenase and  0.25% pancreatin (Sigma-Aldrich, USA) for 10 min. The digestion steps were repeated until the complete digestion of the material. After each digestion, the obtained cells were collected and washed in Dulbecco's medium, as described above. The cell suspensions were pooled and filtered through a 70 μm mesh filter. The cells were suspended in McCoy's 5A medium (Sigma-Aldrich, USA), enriched with 10% horse serum (Sigma-Aldrich, USA) and 2.5% foetal calf serum (Sigma-Aldrich, USA) and seeded onto 6-well culture plates (1.5×10^6 cells/2 ml medium/well). The cells were preincubated for 72 h at 37°C (water-saturated atmosphere, 5% of CO2 and 95% of air). After 48 h of preincubation, 1 ml of fresh, serum-free McCoy's 5A medium was added to each well. After preincubation media were replaced with fresh, enriched McCoy's 5A medium and incubated for another 24 h in the presence of recombinant VIS (100 ng/mL; cat. # 8424-VF, RD System, USA).  Total RNA from APc was isolated using Extrazol (BLIRT, Poland), following the manufacturer's protocol. The purity (A260/A280) and quantity (wavelength 260 nm, A260) of the obtained RNA samples were determined spectrophotometrically using Infinite M200 Pro (Tecan, Männedorf, Switzerland). Bioanalyzer 2100 (Agilent Technology, USA) was used to determine the RNA integrity. Only samples which passed RNA integrity number (RIN) >8 criteria were qualified for the further transcriptome high-throughput sequencing (RNA-Seq) analysis and the quantitative real-time polymerase chain reaction (qPCR) and reverse transcription polymerase chain reaction (RT-PCR) validations. Approximately 5 μg of total RNA was used to remove ribosomal RNA (rRNA) from the samples with the use of  Ribo-Zero Gold rRNA Removal Kit (Illumina, USA). The remaining RNA samples were fragmented  into short fragments using divalent cation buffers at elevated temperature (NEBNext® Magnesium RNA Fragmentation Module, NEB, USA). The obtained, samples were reverse-transcribed to cDNA using SuperScript™II Reverse Transcriptase (Invitrogen, USA). Subsequently, cDNA was used to synthesise U-labeled second-stranded DNA using E. coli DNA polymerase I (NEB, USA), RNase H (NEB, USA) and dUTPSolution (Thermo Fisher, USA). The obtained double-stranded cDNA (dscDNA) was subjected to the end repair and A-tailing processes. At the end, the obtained fragments of dscDNA were ligated with specific adaptors. Quality control analysis and the quantification of the obtained libraries were performed with the use of Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip.