Animals

Animal care and use were conducted in accordance with the guidelines of Experimental Animal Science and Technology Center of Nanchang University (Permit Number: SYXK (gan)-2021–0004, Table S1). Female mice (KM) aged 21 days and 3, 12 and 17 months (3 M, 12 M, 17 M) were housed in specific pathogen-free conditions at the Nanchang University Experimental Animal Center.

Mouse ovary culture in vitro: The ovaries were stripped from 21-day-old female KM mice and cultured as previously described [13]. A 6-well Millicell Standing Inserts (4.0 μm; Millipore Corporate, Billerica, MA; PICM03050) floating on 2 ml Waymouth MB 752/1 culture medium (Sigma, St. Louis, MO) previously equilibrated to 37 °C. Five ovaries were placed on each insert. After 24 h incubation in complete medium, the ovaries were treated with 40 mM NaCl (S8210, Solarbio, Beijing, China, control group, n = 5 mice) or 40 mM succinate (HY-W015410, MedChemexpress, New Jersey, USA, succinate group, n = 5 mice) for 48 h. NaCl and succinate were dissolved in double distilled H2O at an initial concentration and added to culture medium to achieve the desired final concentration. The quality of the ovaries culture was assessed by the pale red, smooth and shiny ovaries under microscope, and pale yellow and clear medium after 48 h incubation with NaCl or succinate.

Mouse ovary administered in vivo: The 3 M KM mice were randomly assigned into the above groups and injected with the corresponding drugs into the ovaries following 2 weeks of feeding. After the 21st day of the administration, the weights of the bodies and ovaries of the mice were measured. Mouse eyeball serum collection: The collected eyeball blood was centrifuged at 12,000 g for 15 min, and the supernatant was collected. The mouse ovaries were photographed with a measuring ruler to compare the size of the ovaries in the two groups.

Induction of mouse POI model: The 3 M KM mice were intraperitoneally administered with 70 mg/kg cytoxan (CTX) (C0768, Sigma-Aldrich, St. Louis, MO, USA) and 12 mg/kg busulfan (BU) (B2635, Sigma-Aldrich) once a week for two weeks according to previous studies [14, 15]. Validation of successful model construction was shown in Fig. S1.

Histological analysis of ovarian tissue and ovarian follicle count

Ovaries were collected and fixed with 4% paraformaldehyde to prepare as paraffin blocks. Sections of 5 μm were obtained and stained with a hematoxylin–eosin (HE) staining kit (AR1180-100, Boster Bioengineering Co., Ltd., Wuhan, China). Digital images were acquired using a microscope (FV3000, Olympus, Tokyo, Japan) and analyzed using ImageJ software (Version 1.52, National Institutes of Health, Bethesda, MD, USA). Follicular stages were determined according to the criterion described previously [16].

Immunohistochemistry (IHC)

IHC was carried out on 5 μm sections of paraffin-embedded tissue. After slides baking, dewaxing, rehydration, the antigen retrieval was performed. The primary antibodies used for IHC were anti-cleaved caspase 3 (#9664, Cell Signaling Technology, Massachusetts, USA) and anti-Ksuc (PTM401, PTMBio, Hangzhou, China) diluted 1:100 in 5% bovine serum albumin (BSA) (SW3015, Solarbio). The secondary antibody and visualized stain for peroxidase was performed using the DAB Substrate Kit (PV-6000D, ZSGBbio, Beijing, China), and slides were counterstained with hematoxylin (AR1180-100, Boster). To confirm the validity and reliability of reagents used in IHC staining especially the specific antibody, the negative control groups were set (Fig. S2A).

Immunofluorescence (IF)

After slides baking, dewaxing, rehydration, the antigen retrieval was performed. The ovarian sections were incubated with 0.1% Triton X-100 for 1 h at room temperature. After blocking with 5% BSA (SW3015, Solarbio) for 1 h, the samples were incubated with the primary antibodies overnight at 4 °C. The primary antibody was anti-pan-Ksuc (1:100, PTM401, PTMBio). The secondary antibody (fluorescein isothiocyanate conjugated goat anti-rabbit IgG, 1:100, E-AB-1014, Elabscience, Wuhan, China) was incubated with the samples for 1 h in the dark at room temperature. The cell nuclei were stained with DAPI Staining Solution (C1005, Beyotime, Shanghai, China). To confirm the validity and reliability of reagents, negative control was set (Fig. S2B). Fluorescence images were obtained using a fluorescence microscope (Olympus, Tokyo, Japan).

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay

Ovarian sections were labeled with TUNEL reagents according to the product brochures (MK1014-100, Boster). The sections were counterstained with hematoxylin to visualize the nucleus. To confirm the validity and reliability of reagents, negative control and positive control were set (Fig. S3). The percentage of TUNEL-positive cells (%) in the mouse ovary was analyzed using ImageJ software.

Western blotting

Total proteins were extracted from mouse ovaries using radio-immunoprecipitation assay (RIPA) buffer (R0010, Solarbio) with phenylmethylsulfonyl fluoride (PMSF) (ST506, Beyotime, Shanghai, China) and protease inhibitor cocktail (HY-K0010, MedChemexpress). Equal amounts (20 μg) of proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Sigma-Aldrich). Subsequently, the membranes were hatched overnight at 4 °C with primary antibodies (Table S2). At room temperature, the horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) was incubated on the membrane for 1 h. Anti-mouse IgG HRP-conjugated goat (SA00001-1) and anti-rabbit IgG HRP-conjugated goat (SA00001-2) secondary antibodies were purchased from Proteintech (Rosemont, IL, USA). Analysis of relative levels of target protein was conducted by quantifying the gray value of the target bands (as for the quantification of Ksuc, representative bands with differences were selected to quantify) normalized to those detected by β-actin using ImageJ software.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated using RNAiso Plus (9108, Takara Bio, Kyoto, Japan). The cDNA was acquired with the PrimeScript RT kit (6210A, Takara Bio, Kyoto, Japan) according to the manufacturer’s instructions. Then, the synthesized cDNA was used as the template for qRT-PCR. Primers are shown in Table S3. The qRT-PCR was performed in triplicate using SYBR Premix Ex TaqTM Kit (MF013, Mei5bio, Beijing, China). Relative expression values were calculated with the 2−ΔΔCt method. Values of gene expression were calculated as the means of three replicates and β-actin was used as a reference.

Hormone assay

Serum estradiol (E2) (E-EL-0150c, Elabscience) and anti-Müllerian hormone (AMH) (E-EL-M3015, Elabscience) levels in the serum were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions.

Statistical methods

All data are represented as mean ± standard error of the mean (SEM) and the data from the experiments were analyzed by one-way analysis of variance (ANOVA) for multiple groups and Student's t-test for two groups with GraphPad Prism 8.0.2. (California, USA).