Animal care and drug administration

Female C57BL/6J mice of 7-week-old were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and fed freely for 1 week for environmental adaptation before drug administration. All the mice were housed in ventilated cages with free access to food and water. The feeding environment was maintained at a temperature of 22 ± 2 °C and a humidity of 55% ± 15% on a 12:12 h light/dark cycle.

The first batch of animal experiments included 2 groups (with 16 female 8-week-old C57BL/6J mice in each group): control group (Con) and doxorubicin group (Dox). Doxorubicin (D8740, Solarbio, China) was intraperitoneally injected once at a dose of 10mg/kg. The second batch of mice were divided into six groups (with 20 female 8-week-old C57BL/6J mice in each group): vehicle control group (Vehicle), doxorubicin group (Dox), doxorubicin plus DQ group (Dox + DQ), doxorubicin plus fisetin group (Dox + F), DQ group, and Fisetin group. DQ (dasatinib, 5 mg/kg; quercetin, 50 mg/kg) or fisetin (100 mg/kg) or vehicle solution (10% polyethylene glycol 400, PEG400) was administered by oral gavage once every other day for 3 weeks [24, 28]. A single intraperitoneal injection of doxorubicin (10 mg/kg) or saline was given at the day after the second administration of senolytics. Senolytic drugs and solvent used in this study are commercially available from MedChemExpress (dasatinib, HY-10181; quercetin, HY-18085; fisetin, HY-N0182; PEG400, HY-Y0873A).

Blood samples and ovaries of female mice were collected at 11 weeks of age. Body weight and ovary weight were recorded. Six ovaries from each group were preserved in 4% paraformaldehyde for subsequent process. Three ovaries from each group were flash-frozen in liquid nitrogen to make frozen sections and the other ovaries were stored at −80 °C for protein or RNA extraction.

Estrous cycle monitoring

Vaginal smears of mice were taken at about 9–11 a.m. every day for consecutive 2 weeks. Vaginal cells procured by washing the vagina with saline were spread on clean glass slides and stained with hematoxylin and eosin (H&E). The stage of the estrous cycle was judged by the cytological characteristics under a microscope as previously described [29]. Mice were categorized as regularly cycling if they consistently displayed cycles that were 4–6 days long. Mice with mean cycle lengths out of this range and mice with cycles of variable lengths or without apparent cycles were categorized as irregularly cycling [30].

Serum hormone measurement

Blood samples of female mice were collected at diestrus. Levels of serum anti-müllerian hormone (AMH), follicle-stimulating hormone (FSH), and estradiol (E2) were measured by enzyme-linked immunosorbent assay according to the manufacturer’s instructions (CSB-E13156m, CSB-06871m, and CSB-E05109m; Cusabio Technology LLC, USA).

Follicle counting

The ovaries fixed in 4% paraformaldehyde were embedded in paraffin and then cut into pieces of 5 μm, with four consecutive pieces mounted on one glass slide. Every fourth slide was stained with H&E and analyzed under a light microscope by 2 persons who were blinded to the group of the sections. Follicles without visible oocytes were excluded while zona pellucida remnants (ZPRs) were classified as late atretic follicles. The detailed procedures were performed as previously described [31].

Mating test

In the second batch of animal experiments, six female mice from each group were randomly selected for mating test. Two female mice and one sexually mature male mouse were kept in a cage for free mating. After 10 days, the female mice were placed in solitary cages. Then, the pregnancy status, parturition outcome, litter size, and pup conditions were monitored and recorded. The mating test was repeated twice. Two rounds of mating test were 50 days apart to make sure all mice were weaned before the second round.

Senescence-associated β-galactosidase (SA-β-gal) staining

SA-β-gal staining of frozen ovarian tissue sections were performed using a commercial SA-β-gal staining kit (C0602, Beyotime Biotechnology, China) according to the manufacture’s protocol. In brief, frozen sections of ovarian tissue were rewarmed at room temperature, soaked with PBS, and then fixed in the fixative solution for 15 min. After washing with PBS for three times, samples were incubated with SA-β-gal working solution at 37 °C for 16 h. Ice-cold PBS was used to stop the enzymatic reaction.

Immunohistochemistry

Three representative ovarian sections in each group were selected for immunohistochemical analysis. After deparaffinization and rehydration, the sections were heated in Tris-Ethylene Diamine Tetraacetic Acid buffer (pH 9.0) for 15 min at 95 °C for antigen retrieval. Next, naturally cooled sections were washed three times with PBS, and incubated in 3% H2O2 solution for 30 min at room temperature. The ovarian sections were blocked in 3% BSA for 1 h and then incubated with primary antibody (p16 #A0262, ABclonal Technology, China; p21 #A11454, ABclonal Technology, China; γH2AX #AP0687, ABclonal Technology, China) overnight at 4 °C. The next day the sections were incubated with secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG (H+L), GB23204, Servicebio, China) for 50 min at room temperature followed by 3 washes with PBS. Subsequently, the sections were visualized with DAB chromogenic agent (AR1022, Boster, China) and counterstained by hematoxylin. Images were acquired using the cellSens Dimension software (Olympus Soft Imaging Solutions GmbH, Germany) under a light microscope and the relative expression was evaluated by Image Pro Plus software [32]. Briefly, we used the straw tool in HSI mode to determine the region for analysis and obtained the integrated optical density (IOD) and area of each image. The mean density was calculated by IOD/Area to represent the corresponding antigen expression level. All images were analyzed under the same analytical environment and color parameters.

Immunofluorescence

The expression levels of typical fibrosis biomarker, alpha-smooth muscle actin (α-SMA), in ovaries were detected by immunofluorescence staining according to the routine procedure [33]. Ovarian sections were incubated with primary antibody (α-SMA #ab124964, Abcam, Cambridge, UK) overnight at 4 °C. The next day, after incubation with secondary antibody (Cy3 conjugated Goat Anti-Rabbit IgG (H+L), GB21303, Servicebio, China) for 60 min at 37 °C, nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (G1012, Servicebio, China). Images were taken by a fluorescence microscope (Olympus).

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

The cell apoptosis in murine ovaries was analyzed using the One Step TUNEL Apoptosis Assay Kit (C1088, Beyotime Biotechnology, China). According to the manufacturer’s instruction, ovarian sections were first permeabilized by Proteinase K at room temperature for 15 min and then incubated with TUNEL reaction mixture at 37 °C for 1 h. The nuclei were counterstained with DAPI. For each sample, 3–5 images were taken from random fields.

Sirius red staining

A series of representative ovarian tissue sections were routinely dewaxed to water and stained with Sirius red dye for 15 min. After dehydration, ovarian sections were sealed with neutral resin and photographed for quantitative analysis.

Western blot

Total protein was extracted from ovaries as the routine procedure and separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins in the gel were subsequently transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skimmed milk for 1 h, membranes were incubated with specific primary antibody (p16 #A11337, ABclonal Technology, China; p21 #PA9426, Abmart, China; and β-actin #AC026, ABclonal Technology, China) overnight at 4 °C. The next day PVDF membranes were washed 3 times with Tris Buffered Saline plus Tween-20 and incubated with appropriate secondary antibody for 1 h at 37 °C. After another 3 washes, target blots were imaged using the chemiluminescence method. Quantification of the protein expression was conducted with Image Lab software based on the integrated light intensity of each band. β-actin expression was measured to verify equal loading.

Quantitative real-time polymerase chain reactions (qRT-PCR)

Total RNA from ovaries was extracted and purified using RNAiso plus reagent (Takara, Nojihigashi, Japan). Samples containing 2 g of total RNA were DNase-treated with gDNA wiper (R223-01, Vazyme, China) and then reverse transcribed into cDNA using HiScript reverse transcriptase (R223-01, Vazyme, China). Real-time PCR was performed on a CFX96 real-time PCR system (Bio-Rad, USA) at a final volume of 40 μl. The Actb gene was used as endogenous control to calculate relative gene expression based on the comparative CT method. The primer sequences are listed in Table 1.

Table 1 The sequences of primers used for qRT-PCR assayStatistical analysis

Data are presented as mean ± SD and a p < 0.05 was considered significantly different. Statistical comparisons were performed using unpaired Student’s t-tests, Chi-square test, or one-way ANOVA. Statistical analyses were performed in SPSS 26.0 and GraphPad Prism 8.0.