Clinical Samples Collection

The study recruited women who accepted oocyte pickup in the Lanzhou University First Affiliated Hospital. The study population comprised the control group (n = 23) and the POI group (n = 25). Inclusion criteria included women aged 18–40 with regular menstrual cycles and no history of ovarian surgery. Exclusion criteria included a history of chemotherapy, radiation therapy, or chronic systemic diseases (e.g., diabetes, autoimmune disorders). Informed consent was gained from all participants. The study was approved by the Ethics Committee of the Lanzhou University First Affiliated Hospital.

Follicular fluid samples were centrifuged at 3000 rpm for 10 min and the upper fluid was stored at -80 ℃.

Cell Culture

KGN cells, a human granulosa cell line, were purchased from Procell (Wuhan, China). The KGN cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin solution, maintained in cell culture incubator at 37 ℃ with 5% CO2.

Mice Treatment

Female C57BL/6 mice (6–8 weeks old) were purchased from Lanzhou Veterinary Research Institute and housed under specific pathogen-free conditions with a temperature of 22 ℃ ± 1 ℃ and humidity of 60% ± 10%. The mice had free access to food and water during rearing. All procedures were approved by the Animal Ethics Committee of the First Hospital of Lanzhou University.

The mice were randomly divided into control group (n = 10) and POI group (n = 10). The mice of POI group were treated with VCD (160 mg/kg) by intraperitoneal injection, and the mice of control group were treated with the same volume of corn oil. The VCD dose (160 mg/kg) was selected based on previous studies demonstrating its efficacy in inducing ovarian insufficiency in mice [22, 23]. The injection was continued for 20 days and then the mice were executed under anesthetic.

Realtime Quantitative Polymerase Chain Reaction (PCR)

Total RNA was extracted by TRIzol reagent (Invitrogen Life Technologies, USA) and reverse transcribed to cDNA by FastKing gDNA Dispelling RT SuperMix kit (TIANGEN BIOTECH, Beijing) following the instruction. The SuperReal PreMix Plus (SYBR Green) kit was used for quantitative PCR amplification. GAPDH was selected as the reference gene.

Enzyme-Linked Immunosorbent Assay (ELISA)

Follicular fluid and mouse serum were collected, centrifuged, and stored at -80 ℃. The levels of ZAR1, FSH, catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured using ELISA kits (FINGHUISHENGWU, China) according to standard protocols.

Cell Transfection

The overexpression plasmid of ZAR1 was obtained from HANBIO (Shanghai, China). LiPOIectamine™ 3000 Reagent kit (Thermo Fisher Scientific Company, USA) was used for transfection. The KGN cells were seeded into 12-well plates (1 × 105cell) and preincubate for 24 h. Each well was washed by PBS. The 2 µL transfection reagent, 5 µL (0.2 µg/µL) plasmid and 50 µL Opti-MEM were mixed and incubated for 15 min at room temperature. Finally, the mixture was added into KGN cells and cultured for 48 h.

Cell Viability Assay

Cell Counting Kit-8 (CCK8; Coolaber, China) was used to measure cell viability. The KGN cells (5 × 103) were seeded in the 96-well plates and treated following experiment scheme. Then we added 10 µL CCK8 and 90 µL serum-free medium to each well. The samples were incubated for 3 h at 37 ℃ and detected with microplate reader (TECAN, Switzerland).

Western Blotting

The protein was extracted from KGN cells and ovary of mice. Proteins (30 µg) were separated by electrophoresis in 10% SDS polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membrane. After blocked with 5% non-fat milk at room temperature for 2 h. Then, the membranes were incubated with primary antibody at 4 ℃ overnight. The primary antibodies including Anti-beta-Actin (1:10000, Bioss), Anti-Bax (1:1000, Bioss), Anti-Bcl-2 (1:1000, Bioss), Anti-caspase-3(1:1000, Bioss), Anti-Cyclin C (1:1000, Bioss), Anti-Cyclin D1 (1:1000, Bioss), Anti-Cyclin E (1:1500, Bioss), Anti-CDK2 (1:800, Bioss), Anti-LC3 (1:1000, Bioss), Anti-p62 (1:2000), Anti-ZAR1 (1:1000, Bioss). Then the membranes were incubated with HRP-conjugated anti-rabbit IgG for 2 h. Finally, the enhanced chemiluminescence (ECL) detection kit (BioRad, USA) was used for proteins detection. The blots were visualized with ChemiDoc XRS + imaging system (Bio-Rad Laboratories, USA) and analyzed with ImageJ.

Apoptosis Detection

Annexin-V PE/7-AAD apoptosis kit (4 A BIOTECH, China) was used for apoptosis detection. KGN cells were seeded into 6-well plate and incubated for 24 h. Then the cells were harvested with trypsin and resuspended with 500 µL 1x binding buffer, then 5 µL PE and 10uL 7-AAD was added into the mixture. Finally, detected the level of apoptosis with flow cytometry (BD, USA).

Cell Cycle Detection

Cell cycle detection kit (4 A BIOTECH, China) was used to detect the changes of cell cycle. KGN cells were seeded into 6-well plate and incubated for 24 h. Then the cells were harvested with trypsin and fixed with 500 µL 70% ethyl alcohol for 2 h. After centrifugation, the 70% ethyl alcohol was removed. Then the sediment was resuspended with 100 µL RNase A and dyed with 400 µL PI for 30 min. Finally, the cell cycle changes were detected with flow cytometry (BD, USA).

ATP Detection

The ATP content detection kit (Solarbio, China) was used for ATP detection. KGN cells (105) were sonicated for 1 min and centrifuged for 10 min at 4 ℃. The supernatant was mixed with 500µL chloroform and centrifuged for 3 min, then extracted the supernatant to detect the ATP content according to the instruction.

Mitochondrial Membrane Potential Detection

The mitochondrial membrane potential detection kit (Beyotime, China) was used for detection of mitochondria damage. First, we seeded 105 KGN cells in the 6-well plate and cultured for 24 h. The 1000x TMRE was diluted to 1x TMRE with buffer solution and added 1mL to each well, then incubated with cells for 30 min at 37 ℃. After washing the cells with serum-free medium we assess the mitochondrial membrane potential level by flow cytometry (BD, USA).

Reactive Oxygen Species (ROS) Detection

The ROS detection kit (Solarbio, China) was used for the assessment of level of ROS. First, we seeded 105 cells in the 6-well plate and cultured for 24 h. Then we diluted DHE dye with serum-free medium (1:1000). The cells were washed with PBS solution and then dyed with the diluted DHE dye for 30 min at 37 ℃. After washing the cells with serum-free medium thrice, we observed the fluorescence intensity under the fluorescence microscope (Olympus, Japan).

Ad-mCherry-GFP-LC3B Transfection

A total of 105 cells were seeded in 12-well plates and cultured for 24 h. Cells were grown on 12-well plates and reached 20–30% confluence at the time of transfection. After washed with PBS, cells were transfected with mCherryGFP-LC3B adenovirus in serum-free medium containing polybrene (5 µg/mL) for 24 h at 37 ℃. Following designed treatment, autophagosome and autophagy flux were observed under fluorescence microscopy (Olympus, Japan).

H&E Staining

The ovaries of mice were fixed in 10% formalin for 24 h, embedded in paraffin and cut into 5 μm thick. After routine deparaffinization, the tissues were stained with hematoxylin for 5 min, flushed for 3 min, and then stained with eosin for 1 min. Following dehydration, the sections were sealed by neutral gum and observed by microscope. Follicle classification was performed according to the Pederson standard [24, 25].

Immunohistochemistry (IHC)

After slicing, drying, hydration and antigen retrieval, the ovary samples were incubated with peroxidase blocker for 30 min at room temperature. Then incubated samples with primary antibodies at 4 ℃ overnight. After rewarming for 1 h, added response enhancement solution to the samples. Then incubated samples with goat-anti-rabbit IgG for 20 min. After DAB dyeing, hematoxylin dyeing and regular dehydration. The samples were sealed and observed under the microscope.

Statistical Analysis

Spearman correlation analysis was performed for the relationship between the level of FSH and the level of ZAR1 in women with or without POI. Comparisons between two groups were performed with T test. Comparisons among three or more groups were performed with one-way analysis of variance (ANOVA). P < 0.05 was considered as statistically significant. All data were analyzed by SPSS 25.0. All experiments were executed at least three times.