Cell Culture and Reagents
The follicular granulosa cells in Hyline-Brown laying hens aged two years were isolated and cultured according to the preliminary research method [15]. In brief, the ovaries of non-gestating laying hens were procured from a local slaughter house and immediately brought to the 4°C refrigerator in the laboratory. The follicular liquid in ovary was extracted by a syringe needle and immediately centrifuged at 800g for 5min to isolate the granulosa cells. Cells were cultured with 0.2% collagenase (abs47048001, Absin, Shanghai, China) in a water bath at 37°C for 6 min. Cells were cultured in high glucose Dulbecco’s Modified Eagle Medium (abs9483, DMEM, Absin, Shanghai, China) containing 14% fetal bovine serum (abs972, Absin, Shanghai, China) at a humidified 5% CO2 incubator (Ou Meng Industrial Co. Ltd., Shanghai, China) at 37°C.
Hg (B8945, purity > 99.0%, Baiaolaibo Bio-Tech, Beijing, China) dissolving in 0.1 M sterile phosphate buffered saline (PBS) was added to the medium to provide 15 μM Hg. Se (sodium selenite, X0367, purity > 99.0%) was obtained from Xinxinjiali Bio-Tech Co. (Wuhan, China) and 4-phenyl butyric acid (B3859, 4-PBA, an inhibitor of ATF6/CHOP pathway) was purchased from Baiaolaibo Bio-Tech Co. (Beijing, China). The Se dissolving in sterile PBS was prepared to the corresponding concentration for the further experiment. Antibodies of CHOP, ATF6 and β-actin were obtained from Abcam (Cambridge, England).
Experimental Design
For detection of the function of ERS in the protection of Se on apoptosis, granulosa cells (GCs) were cultured, exposed with 15μM Hg (15Hg), 12μM Se (12Se) and 0.1μM 4-PBA alone or in combination. The 5 groups were as followed: Control, 15Hg, 12Se, 15Hg +12Se, 15Hg+4-PBA and 15Hg +12Se+4-PBA. The representative pictures of follicular granulosa cells isolated successfully were shown in Supplement Figure 1.
Cell Viability Assay
Cells viability assay was performed according to a cholecystokinin-8 (CCK-8) kit detection. After 24 hours of treatment, the follicular granulosa cells were incubated in 96 well-plates with 10 μL CCK-8 reagent (Absin, Shanghai, China) for 2 hours at 37°C. The absorbance of cells was then measured using a microplate reader (Absin, Shanghai, China) at 450 nm. The cell activity was calculated according to a previous methodology [16].
Nuclear Condensation Assays
When the cells were treated for 24 hours, the follicular granulosa cells fixed in 4% paraformaldehyde were rinsed 3 times with sterile PBS. The cells were incubated with 4',6-diamidino-2-phenylindole (DAPI, Absin, Shanghai, China) to examine the nuclear morphology below a fluorescence microscope (Ou Meng Industrial Co. Ltd., Shanghai, China). The degree of nuclear condensation was qualitatively determined according to fluorescence brightness.
Annexin V/Propidium Iodide Staining Assay
When the cells were treated for 24 hours, the follicular granulosa cells were digested by 0.2% trypsin, the cells suspensions were obtained by means of washing the cells with PBS and centrifugating suspension at 1200g for 5min. The cells were stained with Annexin V-FITC binding and propidum iodide (Absin, Shanghai, China) and quantitated by a flow cytometer (Ou Meng Industrial Co. Ltd., Shanghai, China). The apoptosis rate was calculated as followed: Apoptosis rate (%) = The numbers of early apoptotic and late apoptotic cells / The total number of cells × 100%.
Mitochondrial Membrane Potential Assay
When the cells were treated for 24 hours, the follicular granulosa cells were digested by 0.2% trypsin, the cells suspensions were obtained by means of washing the cells with PBS and centrifugating suspension at 1200g for 5min. Cells were treated with the JC-1 fluorescent probe for 30 minutes at 37°C in a dark environment. The follicular granulosa cells’ fluorescent intensities were determined by a flow cytometer (Ou Meng Industrial Co. Ltd., Shanghai, China). The calculation method of mitochondrial membrane potential reduction is based on a previous study [17].
Quantitative Reverse Transcription-polymerase Chain Reaction
After 24 hours of treatment, the follicular granulosa cells were lysed with cell lysis fluid (Absin, Shanghai, China). The intracellular total RNA was extracted using a TRIzol reagent (Absin, Shanghai, China) and a reverse transcription kit (Absin, Shanghai, China) was used to reverse-transcribed to the cDNA. The PCR assays were carried out in a LightCycler96 (Roche, Basel, Switzerland). The PCR reaction system as as follows: 5 μL of 2 × SYBR Premix Ex TaqTM (Absin, Shanghai, China), 0.5 μL of diluted cDNA template, 4.1 μL of RNase-free water, and forward and reverse primers. The 5’ to 3’ oligonucleotide primer sequences were referenced with a previous literature, which was presented in Supplement Table 1. The PCR amplification was as followed: initial denaturation at 94.5°C for 29 s, followed by 41 cycles of 94.5°C for 5 s, 59.5°C for 29 s, and 71.5°C for 31 s. The 2−ΔΔCt method was used to analyze the relative quantitative data of each gene by the according to a previous study [18].
Western-blot analysis
The expressions of ATF6 and CHOP proteins were determined by the western-blot analysis. Intracellular total proteins were extracted according to the radioimmunoprecipitation method. The total proteins were separated using 10% polyacrylamide gels electrophoresis and then transferred to the polyvinylidene difluoride membranes (Beyotime Co. Ltd, Wuhan, China). The membranes were blocked with 5.0% skim milk for 20 h and incubated overnight with ATF6 (1:500, Beyotime Co. Ltd, Wuhan, China), CHOP (1:500, Beyotime Co. Ltd, Wuhan, China) and β-actin (1:3000, Beyotime Co. LTD, Wuhan, China) antibodies, and then were incubated with the secondary antibody against rabbit IgG. The protein signals were detected using a western blot imaging system (JP-K300, Jinpeng science technology Co. Ltd, Shanghai, China) and relative abundance of each protein was expressed as optical density ratios.
Statistical Analysis
All variance statistical methods in this study were based on one-way variance analysis and all experimental results were expressed as mean ± standard error. Tukey multiple range test was used for the multiple comparison by SPSS 21.0 (SPSS Inc., Chicago, IL, USA) when there was any significant difference among any groups (P < 0.05).