Animals
Eighteen female Sprague-Dawley adult rats (aged 21 days, weight 70-90 g) were purchased from the Laboratory Animal Centre of Wuhan University (No.430727201101344525, Wuhan, China). The rats were allowed to adapt to the environment for two days. All animals were grouped and housed in a room (22± 2 °C, 12-h light/12-h dark cycle) with free access to food and water.
Rats model was induced according to previous studies [21,22].Briefly, rats were randomly divided into PCOS group (n=9) and control group (n=9). The animals in the PCOS group were subcutaneously injected with 6 mg/100 g DHEA (Aladdin Reagent Co., Ltd., Shanghai, China), which was dissolved in olive oil, once a day for 20 consecutive days. The animals in the control group received subcutaneous injections of equivalent olive oil alone.
The procedures involving rats and their care were carried out in accordance with the NIH guidelines (NIH Pub. No. 85-23, revised 1996). The experimenter obtained the certificate of qualification of laboratory animal professional technical examination (W20190028). The study was approved by the Laboratory Animal Welfare & Ethics Committee of Renmin Hospital of Wuhan University (IACUC Issue No. WDRM 20200909).
Sample collection
After the last administration, all the rats fasted for 12 h. Then, all the rats were anaesthetized with isoflurane (5% induction, 1% maintenance) in the early morning of the following day. Immediately after opening the abdominal cavity, for each rat, one ovary was removed and fixed in 4% paraformaldehyde, followed by paraffin embedding, and the other ovary was immediately stored at - 80 °C. Then, the subcutaneous fat and visceral fat were separated. After cutting the leg skin, the skeletal muscles were separated. Two pieces of skeletal muscle, subcutaneous fat and visceral fat, each weighing approximately 2 g, were collected from each animal. One was immediately fixed in 4% paraformaldehyde, followed by paraffin embedding, and the other was immediately stored at - 80 °C. After tissue collection was completed, the chest was opened. Blood was extracted from the heart, placed on ice for 20 minutes, and then separated by centrifugation at 3000 rpm for 20 minutes. The upper layer of serum was collected and stored at - 80 °C.
Serum analysis
According to the manufacturer’s instructions, ENPP1 was detected by enzyme-linked immunosorbent assay (ELISA) kits (JYM1287Ra, Jiyinmei, Wuhan, China). Follicle-stimulating hormone (FSH) (RA20044), luteinizing hormone (LH) (RA20133), testosterone (T) (RA20653), fasting insulin (FINS) (RA20092), adiponectin (ADP) (RA20554), leptin (RA20489), and free fatty acids (FFA) (RA20313) concentrations of serum were detected by ELISA kits (Bioswamp, Wuhan, China). Fasting blood glucose (FBG) (F006-1-1), total cholesterol (TC) (A111-1-1), triglyceride (TG) (A110-1-1), high-density lipoprotein cholesterol (HDL-C) (A112-1-1), and low-density lipoprotein cholesterol (LDL-C) (A113-1-1) were detected by enzymatic colorimetric kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The homeostasis model assessment of IR (HOMA-IR) was calculated using the following formula: HOMA-IR= [FBG (mmol/l) × FINS (mU/l)]/22.5 [23].
Haematoxylin-eosin (H&E) staining
Ovaries, skeletal muscles, subcutaneous fat, and visceral fat were embedded in paraffin, serially sliced into 4-µm-thick sections, and stained with H&E according to the operating instructions (Servicebio, Beijing, China). The above sections were observed under a microscope (NanoZoomer S360, Hamamastu, Japan). ImageJ software (Version 1.53a; NIH, Bethesda, MD, USA) was used to analyse the cross-sectional area (CSA) of skeletal muscles and the mean cell area of subcutaneous fat and visceral fat.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
RNA was extracted from ovaries, skeletal muscle, subcutaneous fat, and visceral fat of rats by TRIzol (Accurate Biology, Changsha, China). Reverse transcription and qRT-PCR were performed using Evo M-MLV RT Mix Kit and qPCR Kit (Accurate Biology, Changsha, China). The specific primers were listed in Table 1. GAPDH served as an internal reference. The amplification conditions were as follows: an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s.
Immunofluorescence (IF) staining
Sections of ovaries were used for IF staining following a previously described protocol [24]. Briefly, paraffin sections were incubated with 10% goat serum in PBS for 30 min to block nonspecific binding of the antibody. Then, the sections were incubated with 1:800 rabbit anti-rat ENPP1 antibody (Bioss, Beijing, China) overnight at 4 °C. Fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG was used as secondary antibody for 2 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Servicebio, Beijing, China) at a dilution of 1:500 for 5 min. An Olympus laser scanning confocal microscope (BX53) was used for observation and photography.
Western blotting
Proteins of ovarian tissues were extracted in radio immunoprecipitation assay (RIPA) buffer in the presence of phosphatase and protease inhibitors. After separation by sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, protein samples were transferred onto polyvinylidene fluoride (PVDF) membranes. After 5% non-fat milk was added, the membranes were blocked at room temperature. The membranes were incubated with 1:2000 rabbit anti-rat ENPP1 antibody (Bioss, Beijing, China) overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Servicebio, Beijing, China) at room temperature for 1 h. The protein bands were visualized by chemiluminescence reagent (Servicebio, Beijing, China) and analysed by ImageJ software.
Statistical analysis
All data were analysed using SPSS 26.0 statistical software (IBM, Armonk, NY, USA) and expressed as the mean ± standard deviation (SD). Differences between the two groups were compared using unpaired t-tests (data with normal distribution) or Mann–Whitney U tests (data with a skewed distribution). Spearman’s rank correlation analysis was used to analyse the rank correlation between data. Software Graphpad Prism version 8.4 (GraphPad Software, San Diego, California) was used to plot bar graphs. Differences between groups with P < 0.05 were considered significant.