Study subjects and sample collection

The diagnosis of PCOS was based on the Rotterdam criteria. Control group comprised women seeking treatment for tubal infertility or male factor issues, who had normal ovarian reserve. Excluded from the study were individuals with conditions such as endometriosis, ovarian cancer, or other endocrine disorders. Follicular fluid (FF) samples were collected from 18 PCOS patients and 22 controls for cytokine analysis. Clinical characteristics of the participants were detailed in Table 1. FF samples were immediately centrifuged at 550 ×g for 15 min, and the resulting supernatants were stored at -80 °C until further analysis. GCs were isolated through Percoll gradient centrifugation [17].

Data analysis

The microarray datasets GSE34562, GSE98421, GSE216609, and GSE106724 were retrieved from the GEO database. To ensure data compatibility, raw data were processed using the GEO2R tool (https://www.ncbi.nlm.nih.gov/geo/geo2r/). Bioinformatics tools like the STRING database (https://string-db.org/) were used. Relevant genes (such as GSDME and IL2RG) were input and a protein-protein interaction network, which demonstrates the interaction relationships among gene products, was plotted. Data of GSDME (also called DFNA5) and IL2RG genes in various cancer types (https://compbio.cn/timer2) were retrieved. Spearman’s correlation analysis was conducted, significance by P-value was determined, and results with color-coding and symbols were visually represented. DFNA5 (GSDME) and IL2RG data from GEPIA (http://gepia.cancer-pku.cn/detail.php?clicktag = correlation) were extracted and Spearman’s correlation analysis to show their relationship was performed.

PCOS modeling

Sprague-Dawley female rats (6–8 weeks old) were obtained from Vital River Laboratory Animal Technology Co Ltd. (Beijing, China). IL2RG knockout (KO) rats were sourced from Biocytogen (Beijing, China). The IL2RG KO rats were generated by Biocytogen using CRISPR/Cas9 technology to target exon 2 of the IL2RG gene. By removing a portion of exon 2 (Δ14/Δ14), the normal Il2rg sequence is disrupted, resulting in a non-functional protein and creating an immunodeficient rat model suitable for studying immune-related diseases and humanized research applications. The rats were housed in pairs per cage under a 12-h light/dark cycle, with controlled temperature and humidity. The rats were randomly assigned to four groups: wild type (WT) rats treated with corn oil (WT + C), WT rats treated with dehydroepiandrosterone (DHEA) (WT + D), KO rats treated with corn oil (KO + C), and KO rats treated with DHEA (KO + D) (n = 7–8 per group). To establish the PCOS model, we administered DHEA subcutaneously, as previously described in the literature [18, 19]. This model is characterized by elevated androgen levels, increased LH/FSH ratio, cystic follicle development, and disrupted estrous cycles, mimicking key features of human PCOS [20]. To induce the PCOS phenotype, rats in the DHEA group were subcutaneously injected with 6 mg/100 g bodyweight DHEA (CAS. 53-43-0, Aladdin, Shanghai, China) daily for 21 consecutive days. Rats in the control group received the same volume of corn oil injections.

Histology

Five litters per group and one rat per litter were randomly selected for histological analysis. The ovaries were fixed in Bouin’s fluid and then sectioned into 5 μm thick slices for histological examination. All sections were stained with hematoxylin and eosin (H&E). Immunohistochemical (IHC) staining for IL2RG was performed according to the manufacturer’s instructions [21]. The sections were incubated overnight at 4 °C in a humid chamber with a rabbit antibody against IL2RG (ab273023, Abcam, USA) diluted at a ratio of 1:100. Observations were conducted using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). The slides were digitally scanned using the Nanozoomer-XR scanner (Hamamatsu, Japan) at a scanning speed of 0.23 μm/pixel to obtain digital images.

Serum hormone ELISA measurement

Serum level of estradiol (E2) and total testosterone (TT) were measured by an Immunoassay Analyzer using Immulite2000 E2 kit (Sinopharm Group Medical Supply Chain Services Co, Hangzhou, China) and Immulite2000 testosterone kit, respectively. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), and sex hormone-binding globulin (SHBG) levels were all determined using commercial human-specific ELISA kits (Solarbio, Beijing, China) following the manufacturer’s instructions. Free androgen index (FAI) = TT/SHBG*100%.

Glucose tolerance tests

Rats were fasted for 12 h before undergoing glucose tolerance tests (GTT). Glucose levels were measured by tail vein blood sampling using the AccuChek Performa blood glucose analyzer (Roche Diagnostics, Indianapolis, IN, USA). The rats were intraperitoneally injected with D-glucose (2 g/kg body weight) for GTT after measuring glucose levels, and tail samples were collected at 0, 15, 30, 60, 90, and 120 min post-injection for glucose level detection.

Estrous cycle monitoring

Typically, a regular reproductive cycle in female rats consists of four distinct phases (proestrus, estrus, metestrus, and diestrus), which can be identified by examining the cellular composition in vaginal smears. We collected vaginal samples from all mice participants and employed Gram Staining (Solarbio, China) to analyze their estrous cycles under a microscope (Nikon Eclipse 80i, Tokyo, Japan).

Western blot analysis

Three ovaries were taken from each of three randomly selected litters in each group for western blot analysis. The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, China). The protein membranes were blocked with skim milk in TBS-T for 1 h and incubated overnight at 4 °C with the primary antibody in blocking buffer. The membrane was incubated by following antigens: anti-CASP3 [9662, Cell Signaling Technology (CST), Danvers, MA, USA, 1:1000), anti-IL2RG (ab273023, Abcam, 1:1000), β-Actin (ACTB, 4970, CST, 1:1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AF7021, Affinity Biologicals, Ancaster, Ontario, Canada, 1:1000). The membrane was then incubated with HRP-conjugated anti-rabbit IgG secondary antibody (GAR007, Multi Sciences, Hangzhou, China, 1:2000) for 2 h and chemiluminescence images were developed using a Thermo Scientific Pierce ECL chemiluminescence kit (Pierce, USA). The gray scale intensity of the proteins was calculated using Bio-Rad Image Lab software (Hercules, CA, USA).

Cell culture and treatment

KGN (human ovarian granulosa cell tumor) cell line which is a good model for studying GC biology [22] was obtained from Fuheng Biology (Shanghai, China) and cells were cultured in DMEM/F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO2. To evaluate the effects of hyperandrogenism on KGN cells, cells were treated with 10 µM testosterone (T) for 24 hours. siRNA against IL2RG (siIL2RG) and its negative control were transfected using Lipofectamine 3000 (Life Technologies, Gaithersburg, MD, USA). The target sequences of the siRNAs were as follows: siIL2RG-1: forward: 5’-GGAACAACAGAUUCUUGAATT-3’ and reverse: 5’-UUCAAGAAUCUGUUGUUCCTT-3’; siIL2RG-2: forward: 5’-GGUACAAGAACUCGGAUAATT-3’ and reverse: 5’-UUAUCCGAGUUCUUGUACCTT-3’; siIL2RG-3: forward: 5’-CCUGGAGUGGUGUGUCUAATT-3’ and reverse: 5’-UUAGACACACCACUCCAGGTT-3’. CASP3 inhibitor, Z-DEVD-FMK (ZDF, GlpBio, Montclair, CA, USA), was treated at a concentration of 20 µM. Caspase-1 inhibitor, Belnacasan (VX-765, MCE, New Jersey, USA), was treated at a concentration of 10 µM.

Scanning electron microscopy (SEM)

The KGN cells were first fixed in 2.5% glutaraldehyde and then dehydrated through a gradient series of ethanol solutions (30%, 50%, 70%, 100%, and 100%) for 10 min each. After dehydration, the samples were transferred to a supercritical dryer for critical point drying. Once the drying process was complete, the samples were removed from the dryer, affixed to a sample table with conductive tape, coated with gold, and examined using a scanning electron microscope (SU8020, Hitachi, Japan). For conventional sample preparation, the samples were directly affixed to the sample table with conductive tape, coated with gold, and examined.

Quantitative Real-Time polymerase chain reaction (qRT-PCR)

The levels of specific mRNAs (IL2RG) in KGN cells and the internal control mRNA ACTB and positive control GAPDH were examined using qRT-PCR. Ovarian tissue was subjected to RNA extraction using a Trizol kit (Vazyme, Nanjing, China), following a previously established protocol [21]. Reverse transcription was performed using a reverse transcription kit and random primers (Vazyme). qRT-PCR was conducted using the SYBR Green qPCR kit (Bio Smile Co., China). The Ct values from each qRT-PCR run were recorded and used to generate a standard equation for the standards. The relative mRNA levels were normalized to ACTB for further analysis. Specific sequence primers were designed as follows: IL2RG: forward: 5’-GTGCAGCCACTATCTATTCTCTG-3’ and reverse: 5’-GTGAAGTGTTAGGTTCTCTGGAG-3’; ACTB: forward: 5’-CTCCATCCTGGCCTCGCTGT-3’ and reverse: 5’-GCTGTCACCTTCACCGTTCC-3’; GAPDH: forward: 5’-CTGCACCACCAACTGCTT-3’ and reverse: 5’-TTCTGGGTGGCAGTGATG-3’.

Immunofluorescence

The death pattern was accurately characterized by adding propidium iodide (PI) and Fluorescein isothiocyanate (FITC)-Annexin V to the culture medium, PI is commonly used in flow cytometry and fluorescence microscopy to identify dead cells. Since it cannot pass through an intact cell membrane, it only stains cells with compromised membranes (such as dead or dying cells), allowing for the discrimination between live and dead cells in a sample. PI and FITC-Annexin V was gently mixed to avoid damaging lytic cell death. Cells were labeled with Annexin V and FITC-Annexin V (AP101, Lianke, China) and assessed at 0 to 24 h. Images were captured using the Operetta CLS™ high-content analysis system (UltraVIEW VOX, PerkinElmer, USA). At least three independent fields were imaged.

Lactate dehydrogenase (LDH) assay

KGN cells were cultured in 96-well plates at 1 × 10⁴ cells/well for 24 h. The cell culture supernatants were detected using an LDH Cytotoxicity Assay Kit (C0016, Beyotime, China), following the manufacturer’s instructions. All groups were replicated three times.

Flow cytometry

The cells were seeded at a density of approximately 70% before drug treatments. After harvesting, the cells were washed with cold phosphate buffered saline (PBS) and stained with FITC-labeled Annexin V and PI using the FITC Annexin V apoptosis kit (AP101, Lianke, China). The data were obtained using the CytoFLEX (Beckman Coulter) and analyzed with CytExpert software.

Statistics

Assays were repeated three to four times. Data are presented as means ± SEM (standard error of the mean). Student’s t-test of two independent samples was used for data with normal distribution. Shapiro-Wilk test was used to test normality of small-size samples. When the data showed normal distribution, Pearson’s correlation analysis was used for correlation analysis. When the data did not show normal distribution, non-parametric test was used. The one-way ANOVA with Tukey’s multiple comparison post-hoc test was used for multiple groups. The post hoc statistical power value is expressed as “$”. Significant differences are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.