C57BL6/J mice (4-week-old) were purchased from Charles River Laboratories. The animals were housed in cages with a 12:12 h light:dark cycle set at ambient temperature (22°C-25°C). After acclimation of ordinary food for one-week, female mice were fed either chow diet (Research Diets, D12450J) or high-fat diet (HFD) (Research Diets, D12492) for 12 weeks respectively. Caloric composition of chow diet consisted of 20% protein, 10% fat and 70% carbohydrates, while high-fat diet had 20% protein, 60% fat and 20% carbohydrates. The mice were weighed weekly. After 12 weeks, the female mice were mated with fertile males of the same strain, which were fed with normal diet. The appearance of a vaginal plug was marked as day 1 of pregnancy (D1). The serum and tissue samples on D7 were collected and stored for further studies. All animal procedures were approved by the Ethics Committee of Chongqing Medical University, China.
The human ovarian granulosa cell line KGN was purchased from EK-Bioscience (Shanghai, China) and authenticated through STR analysis by Cellcook Biotech (Guangzhou, China). The cells were cultured at 37°C in DMEM/F-12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Beyotime, Shanghai, China). In order to establish the high-fat cell model, we treated the cells with 400μM Oleate acid (OA, Sigma, USA) and 200μM Palmitic acid (PA, Sigma, USA) for 24h. After that, luteinization was induced in the cells with 1IU human Chorionic Gonadotropin (hCG, Merck, Germany) for 12h.
Real time PCR
Total RNA was extracted from the ovaries and KGN cells with the Trizol reagent (Invitrogen, USA). cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time RT-PCR reactions were performed using GoTaq® qPCR Master Mix Technical Manual (Promega, USA) and ABI ViiATM7 real-time fluorescence quantitative PCR instrument. Data was analyzed using the ΔΔCt method; and were normalized to β-actin. Primer sequences are listed in Supplemental Table 1 and 2.
Tissues and cells were homogenized in RIPA buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitor cocktail (Thermo Fisher, USA). The BCA kit (Beyotime, Shanghai, China) was used to measure the protein concentration. Equal amounts of proteins were separated using 10% SDS-PAGE and then transferred onto a PVDF membrane. The nonspecific binding sites on the membranes were blocked with 5% non-fat milk at room temperature for 1 h. Then, the membranes were incubated with primary antibodies (Supplemental Table 3) at 4 °C overnight. After washing with PBST, the membranes were incubated with anti-rabbit or anti-mouse IgG secondary antibody conjugated with horseradish peroxidase. Finally, the bands on the membranes were visualized using an ECL detection kit (Millipore, Germany). The light density and grey level were analyzed and calculated by using the ImageJ software. β-actin was used as a loading control.
Blood was centrifugated at 3000 rpm for 10 min under 4 °C to obtain serum. Serum levels of triglyceride (TG) and total cholesterol (TC) were determined using specific assay kits (Nanjing Jiancheng, Nanjing, China) through glycerol-3-phosphate oxidase p-aminophenol (GPO-PAP) and cholesterol oxidase-peroxidase coupling (COD-PAP) methods. Ovarian tissue and KGN cells were homogenated with absolute ethyl alcohol over ice, then TG was tested according to Triglyceride test kit (Nanjing Jiancheng, Nanjing, China) protocols, by GPO-PAP method. TG level in tissues and cells were expressed as mol/g protein. Protein concentration was detected with the BCA protein assay kit.
Serum and cell medium estradiol and progesterone levels were determined using commercial enzyme-linked immunosorbent assay kits (ELISA, Yanhui Biotech, Shanghai, China) according to the manufacturer’s protocols. Absorbance was read at 450 nm.
Hematoxylin and eosin (H&E) staining
Ovarian tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Embedded tissues were sectioned at 5μm and stained with H&E prior to the assessment of the morphological changes in the condition of high-fat diet.
Paraffin-embedded sections (5μm thickness) were deparaffinized and treated with citrate repair solution in microwave oven for 5 min at high fire and 15 min at low fire. After repairing, the rabbit SP kit for immunohistochemistry (ZSGB-BIO, Beijing, China) was used for the subsequent procedures. Firstly, endogenous peroxidases were inhibited via incubation with 3% hydrogen peroxide for 10 min at room temperature. Secondly, the nonspecific binding sites were blocked with 10% goat serum for 30 min at 37°C and then incubated with primary antibodies (Supplemental Table 3) overnight at 4℃. Then, the sections were incubated with horseradish peroxidase-conjugated secondary antibody for 30 min at 37°C. Finally, peroxidase activity was detected using the 3,3-diaminobenzidine substrate (DAB, ZSGB-BIO, Beijing, China). The sections were counterstained with hematoxylin. Positive area quantification was calculated by using ImageJ.
Transmission electron microscopy
Specimens were fixed in 2.5% glutaraldehyde, dehydrated in graded alcohol and stained with OsO4 and uranyl acetate. The sections were polymerized and cut to prepare for use on a Hitachi H-7650 transmission electron microscope.
Oil Red O staining
Ovarian tissues were fixed in 4% paraformaldehyde for 24 h and 30% sucrose for 1 h, then embedded in OCT and serially sectioned at 4μm. KGN cells were cultured on 13mm round glass coverslips. After incubating in 60% isopropyl alcohol for 5 min, both sections and cells were stained with freshly prepared 0.15% Oil Red O (Solarbio, Beijing, China) for 10 min. Then, sections and cells were washed in 60% isopropyl alcohol to remove unspecific attachments. Finally, the sections and cells were counterstained with hematoxylin and mounted with glycerol jelly.
Ovarian tissue was homogenized on ice, centrifugation at 12000g at 4℃ for 5 min and the supernatant taken for lactate detection. For KGN cells, culture medium was directly used for lactate detection. Lactate content was determined by a lactate test kit (Nanjing Jiancheng, Nanjing, China) according to the instructions of the manufacturer. Briefly, 1 ml enzyme reagent, and 0.2 ml Color-substrate solution were added to the samples, mix well and incubated in 37 ° C water bath for 10 min. 2 ml stop buffer was then added and the absorbance measured at 530nm. The lactate content was then normalized to total protein content.
ATP levels were measured using ATP Assay Kit (Beyotime, Shanghai, China). Tissue and cells were homogenized on ice, centrifugation at 12000g at 4℃ for 5 min and the supernatant taken. ATP detection reagent add to the supernatant and measured with luminometer at 10 s RLU and concentration calculated based on an ATP standard curve and expressed as mol/mg. Protein concentration was determined using the BCA protein assay kit.
KGN cells were seeded on a 96-well microtiter plate (5.0×103 cells/well) and the Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) was used to determine the number of viable cells. The CCK-8 reagent was added to each well and incubated for 2 h. Then, the absorbance of each well was measured at 450 nm.
Differences between two groups were determined using unpaired Student’s t-test (2-tailed). Differences among three groups were calculated using one-way analysis of variance (ANOVA), while differences within groups were assessed using the Mann-Whitney test. All data are shown as mean ± standard error of the mean (SEM). GraphPad Prism version 5.4 was used for all statistical analyses, and differences were considered significant when P < 0.05. All experiments were repeated, at least, 3 times.