Reagents and antibodies
ZEA (Z2125) was purchased from Sigma-Aldrich (St. Louis, MO, USA)；Pregnant mare serum gonadotropin (PMSG) from Shu Sheng Hormone (Ningbo, China); Dulbecco’s modified Eagles medium with Hams F-12 nutrient mixture (DMEM/F12, 1:1) and fetal bovine serum (FBS) were obtained from Biological Industries (Israel)；the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were purchased from Solarbio Life Science (Beijing, China). Cell cycle detection kit (using PI/RNase A) was obtained from Keygen Biotechnology (Jiangsu, China)；cleaved-caspase-3 (ab214430), Bax (ab32503) and Bcl-2 (ab182858) antibodies were from Abcam (Cambridge, MA, USA), and cleaved-PARP (94885) was purchased from Cell Signaling Technology (Boston, MA, USA)。
Scu shown in Fig 1 was obtained from National institutes for Food and Drug Control (Beijing, China), and the purity was 91.7%.
Isolation and culture of mouse GCs
The mouse GCs were collected and cultured according to our previously describe study25. Briefly, three-week-old female Kunming mice were intraperitoneally injected with 5 IU PMSG, and were euthanized 46 h later. Bilateral ovaries were collected and punctured with 26 gauged# needles under a stereomicroscope to isolate GCs and passed through 0.074 mm sieve and centrifuged (1000 rpm, 5 min). After washing three times with PBS, the cells were resuspended in DMEM/F12 medium supplemented with 10% FBS and 1% streptomycin-penicillin, and were incubated at 37°C with 5% CO2.
Determination of cell viability
Isolated cells were seeded in 96-well plates at a density of 1×105cells/mL. After 24 h of culture, the cells were treated with different concentrations (2000, 1000, 500, 250, 125, 62.5 and 31.25 μg / mL) of Scu for 24 and 48 h; or treated with different concentrations of ZEA (10, 30, 60, 90 and 120 μM) for 24 h; or treated with 60 μM ZEA for different time points (6, 12, 24, 36 and 48 h); or treated with combinations of different concentrations (2000, 1000, 500 μg / mL) of Scu with ZEA (60 μM) for 24 h. Cell viability was detected using MTT assay. Briefly, the medium was discarded, and cells were incubated with 25 μL MTT at 37°C for 4 h. 150 μL DMSO was added to dissolve the formazan crystals at 37°C for 30 min. The OD490 values were obtained using a microplate reader.
Cell cycle distribution analysis
Cells were seeded in 6-well plates at a density of 1×106cells/mL. After reaching 80-90% confluency, the cells were treated with the medium containing different concentrations of Scu (2000, 1000 and 500 μg/mL) with or without ZEA (60 μM) for 24 h. Cells were collected and fixed with cold 70% ethanol for 2 h. After being washed with PBS, cells were incubated with PI/RNase A in dark for 30 min, then the cell cycle distribution was detected using flow cytometry.
Animals and Treatments
Five-week-old female Kunming mice were provided by Charles River (Beijing, China), and were housed under standard laboratory conditions of room temperature (22-24°C) and relative humidity (50-60%), with a 12 h light-dark cycle. The mice were allowed free access to full rodent food and water. The overview of the experimental protocol is shown in Fig 2. The mice were allowed to acclimatize for 1 week. The mice were then randomly divided into control, model, scutellarin groups with eight mice in each group. The model and scutellarin groups were both treated with a single intragastric administration of ZEA dissolved in corn oil at 40 mg/kg. After 2 h, Scu group was intragastrically given 100 mg/kg Scu in PBS (PH=7.4) for 3 days. The control group was intragastric administered with corn oil and then 2 h later with PBS (Fig.2). All mice were weighed and sacrificed post 72 h of ZEA administration, and the ovaries were collected for further study. These procedures in the protocol was performed by conforming the regulations and guidelines of ethical committee of Shanxi Agricultural University (Taigu, China).
In situ TUNEL fluorescence staining assay
Apoptotic cells were detected using deadendTM fluorometric TUNEL system according to the manufacturer’s protocol (Promega, Germany). Briefly, after treated with medium containing different concentrations of Scu (2000, 1000, and 500 μg/mL) with ZEA (60 μM) for 24 h, the cells were fixed with 4% paraformaldehyde at 4℃ for 25 min. After two washes with PBS, the cells were incubated with 0.2% Triton X-100 for 5 min. After washing, cells were equilibrated with 100 µL equilibration buffers at room temperature for 8 min. The equilibration buffer was discarded and 50 µL rTdT incubation buffer was added to the cells on a 5cm2 area on a tissue slide, and incubated at 37°C for 60 min. The cells were then incubated with 2×SSC for 15 min. After being washed, the slides were sealed with mounting medium with DAPI, and analyzed under a fluorescence microscope.
Apoptosis in the ovarian tissues was also investigated by TUNEL staining. Briefly, 4% paraformaldehyde-fixed, paraffin-embedded sections were deparaffinized, rehydrated, treated with 20 μg/mL proteinase K for 8 min at room temperature, and then re-fixed with 4% paraformaldehyde for 5 min. After washing, the sections were treated with 100 µL equilibration buffers at room temperature for 8 min, and then incubated with rTdT incubation buffer at 37°C for 1 h in a humidified chamber away from light. After reaction, the sections were washed with 2×SSC for 15 min. Finally, the sections were sealed with mounting medium with DAPI, and analyzed under a fluorescence microscope.
Western blotting analysis
Total proteins were extracted from cells or tissues using a total protein extraction kit (KeyGen, China), and protein concentrations were measured using BCA assay. Proteins were separated via SDS-PAGE and then transferred to a PVDF membrane. After being blocked with 5% non-fat dry milk for 2 h at room temperature, the membranes were subsequently incubated with primary antibodies to β-actin, Bax, Bcl-2, cleaved-caspase-3 or cleaved-PARP) 4°C overnight. After three washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After three final washes with TBST, the protein bands were observed using an ECL kit (CWBIO, China), and the band intensities were analyzed using Image J software.
Immunohistochemical staining was conducted according to the manufacturers’ instructions. Briefly, 4% paraformaldehyde-fixed, paraffin-embedded sections were deparaffinized, rehydrated, and treated with H2O2 for 10 min. After washing with PBS, the sections were blocked with 5% BSA for 10 min. Then 40 μL cleaved-caspase-3 antibody (1:50) was added to each section and incubated at 4°C overnight. After rinsing with PBS, the sections were incubated with a biotinylated secondary antibody for 10 min at room temperature. After being washed, the sections were added the streptavidin labeled with catalase. Subsequently, the DAB was used for color development. The sections were stained with hematoxylin for tissue morphology. After being dehydrated with gradient ethanol and cleared with xylene, the sections were sealed with neutral resin. The signals were observed under a fluorescence microscope and photographed.
The data were presented as mean ±standard deviation (SD). All statistical analysis procedures were performed in GraphPad PrismTM 5 software (GraphPad Software Inc., La Jolla, CA, USA), and one-way analysis of variance (ANOVA) was used to determine significant differences among groups. *p<0.05; **p<0.01; ***p<0.001.