For the control group, ovarian were collected from female mice in the diestrus phase as described above. For gonadotropin induction ovarian, female mice were injected with 5 IU pregnant mare serum gonadotropin (PMSG) (Sansheng Pharmaceuticals, Ningbo, China), followed by 5 IU human chorionic gonadotropin (hCG) (Sansheng Pharmaceutical, Ningbo, China) 48 h later. Finally, ovarian tissues were collected after 4 h and put into 4% paraformaldehyde (158,127, Sigma, St. Louis, MO, USA) in PBS to fix. The method of preparing slices is described above. Then ovarian slides were deparaffinized, rehydrated, and disposed in Tris-EDTA buffer (pH 9.0). The sections were blocked with 5% goat serum and incubated with primary antibody against HAT1 (Rabbit / IgG, 1:200, Unconjugated, 11432-1-AP, Proteintech, China) at 4℃ overnight. Subsequently, the sections were incubated with a goat anti-rabbit secondary antibody (Goat / IgG, HRP-conjugated, PV-6001, ZSGB-BIO, Beijing, China) for 45 min at room temperature and followed by staining with a DAB peroxidase substrate kit (ZSGB-BIO, Beijing, China).

Extraction of RNA and quantitative real-time PCR (qRT-PCR)

The ovaries were collected from 6 weeks and 10 months old ICR mice and washed with precooled saline to remove blood. 500 µL TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to each ovary in a 1.5 mL tube. Then they were treated with 60 W ultrasound for 3 s with 5 s off by an Ultrasonic cell grinder (Scientz-IID, Xinzhi, China). The entire operation was on the ice and lasted for 1 min. For KGN cells, after they were cultured with stimulation, the medium was discarded and the cells were washed twice with 1–2 mL precooled PBS. An appropriate amount of TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) (500 µL per well in 6-well plate) was added, blown several times with a pipetting gun, transferred to RNA-specific enzyme-free EP tubes, vortexed and mixed, and left for 10 min at room temperature. The next steps were the same for both ovaries and cells. We added chloroform of 1/5 volume of TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) in tubes, vortexed and mixed, and left for 10 min at room temperature. The samples were centrifuged at 15000 rpm for 10 min at 4℃, the supernatant was poured off, and 1 mL precooled 70% ethanol was added. The samples were centrifuged at 15000 rpm for 10 min at 4℃ again. The supernatant was carefully sucked off. 20 µL of DEPC water was added to measure RNA concentration. Then RNA was transcribed to cDNA by 5× All-In-One RT MasterMix (Vazyme, Jiangsu, China). Each reaction system included up to 2 µg RNA template, 2 µl AccuRT Reaction Mix (4×), and up to a total volume of 8 µl nuclease-free H2O. Then incubated at room temperature for 5 min and added 2 µl AccuRT Reaction Stopper (5×), 4 µl 5×All-In-One RT MasterMix, and 6 µl nuclease-free H2O. PCR was performed after thoroughly mixing with the amplification conditions as follows: 25℃ for 10 min, 42℃ for 15 min, and 85℃ for 5 min. After the reaction, the qRT-PCR started, containing 10 µl of SYBR-Green Mixture, 0.5 µl of Primer-F, 0.5 µl of Primer-R, 2 µl of cDNA, and 7 µl of ddH2O. The following primer sequences were used: HAT1 (mouse): forward, 5’-TCTAGCTTCGCCTAGCTTCC-3’, reverse, 5’-GCAACTACTTGGCACAACCA-3’; HAT1 (human): forward, 5’-GTGCAGTGGCATGATTGCGG-3’, reverse, 5’-CACTTTGGGAGGCCAAGGCA-3’; 18 S (mouse): forward, 5’-ATGGCCGTTCTTAGTTGGTG-3’, reverse, 5’-CGGACATCTAAGGGCATCAC-3’; 18 S (human): forward, 5’-CGGCTACCACATCCAAGGAA-3’, reverse, 5’-CTGGAATTACCGCGGCT-3’. All the data were normalized to the expression of 18 S using the comparative 2−ΔΔCt method.

Western blotting

Total proteins from ovarian tissues and cultured KGN cells were extracted using Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) containing protease inhibitors and treated with ultrasound. Then we put both ovarian tissues and KGN cells at 4℃ for 30 min with rotation. After centrifuging at 12,000 rpm for 5 min, the supernatant was collected and quantified by a BCA assay kit (Beyotime, China). We performed western blotting with the same amounts of protein. It is worth mentioning that the samples in Fig. 2E and 5A were extracted from the same batch of KGN cells, which resulted in the same loading control. Then the proteins were loaded onto 10% SDS-PAGE gels, separated by it, and transferred to polyvinylidene difluoride (PVDF) membranes (Sigma, St. Louis, MO, USA). At room temperature, the blots were blocked in 5% (w/v) nonfat milk in TBST for 1 h and incubated with the following primary antibodies at 4℃ overnight: anti-HAT1 antibody (Rabbit / IgG, 1:1000, Unconjugated, 11432-1-AP, Proteintech, China), anti-Caspase 3 antibody (Rabbit / IgG, 1:1000, Unconjugated, 9662s, CST, USA), anti-Cleaved caspase 3 antibody (Rabbit / IgG, 1:1000, Unconjugated, 9661s, CST, USA), anti-Bax antibody (Rabbit / IgG, 1:1000, Unconjugated, ab32503, Sigma, St. Louis, MO, USA), anti-Bcl2 antibody (Rabbit / IgG, 1:1000, Unconjugated, 12789-1-AP, Proteintech, China), anti-β-actin antibody (Rabbit / IgG, 1:10000, Unconjugated, P30002M, Abmart, China), anti-AREG antibody (Mouse / IgG, 1:500, Unconjugated, sc-74,501, Santa Cruz Biotechnology), anti-Foxhead box transcription factor O1 (FoxO1) antibody (Mouse / IgG, 1:1000, Unconjugated, mb0093, Bioworld), anti-P-FoxO1 antibody (Rabbit / IgG, 1:1000, Unconjugated, CY6217, Abways), anti-Ac-FoxO1 antibody (Rabbit / IgG, 1:1000, Unconjugated, AF2305, Affinity), and anti-Lamin B1 antibody (Rabbit / IgG, 1:1000, Unconjugated, 12987-1-AP, Proteintech, China). The membranes were incubated by HRP-conjugated secondary antibodies (Goat / IgG, 1:10000, ZB-2301, HRP conjugated, Zsbio, China) at room temperature for 1 h after washing three times. Finally, the bands were detected using chemiluminescence (ECL) reagents. The Mean Grey Value of the target proteins was estimated by ImageJ software (NIH, Bethesda, MD, USA).

DOs and COCs collection and in vitro maturation (IVM) culture

The ovaries collected from 3-week-old ICR female mice were cut with a blade and soaked in M2 medium (Sigma, St. Louis, MO, USA). DOs in the GV stage were obtained. COCs were isolated from the antral follicles using a disposable syringe with a 20-gauge needle. After the collection of DOs and COCs, we transferred them into MEMα maturation medium covered with liquid paraffin oil in an incubator at 37 °C under 5% CO2. The germinal vesicle breakdown (GVBD) and PBE rate were counted after IVM of 4 and 14 h. The MEMα maturation medium contains 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 10ng/mL EGF (Sigma, St. Louis, MO, USA), and 1.5 IU/mL hCG (Sansheng Pharmaceutical, Ningbo, China). The dose of AA used to culture DOs in this study was 40 µM and the dose of AA used to culture COCs was 10 µM, 20 µM, and 40 µM respectively. Moreover, oocytes used in the following experiments including oocyte immunofluorescence, chromosome spread, in vitro fertilization (IVF), and embryo culture were from COCs of the control group and 40 µM AA-treatment group after IVM of 14 h. The details are described below. All oocyte-related procedures were performed under a stereoscopic microscope (Nikon, Shanghai, China).

Cell culture

Human granulosa cell (GC)-like line KGN cells were cultured with DMEM/F12 (Gibco, Grand Island, NY, USA) containing 10% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere containing 5% CO2.

Small interfering RNA transfection

To knock down HAT1 expression, KGN cells were cultured in 6-well plates and COCs were cultured at MEMα maturation medium. Then they were both transfected with small interfering RNAs (siRNAs) targeting HAT1 (si-HAT1) using Lipofectamine™ 3000 (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. The siRNAs sequences for HAT1 (mouse) and HAT1 (human) were 5’-CCGGGAAAGATTACTGCAA-3’ and 5’-GGAAGATTACCGGCGTGTT-3’ respectively.

Oocyte immunofluorescence

Oocytes were fixed in PBS-buffered 4% paraformaldehyde (Sigma, St. Louis, MO, USA) for 30 min, followed by permeabilization with 0.5% Triton X-100 (Sigma, St. Louis, MO, USA) for 20 min. The oocytes were washed three times and blocked in 1% BSA for 1 h. After incubation with anti-α-tubulin (Mouse / IgG, 1:200, FITC conjugate, F2168, Sigma, St. Louis, MO, USA) at 4℃ overnight, the oocytes were washed three times again as said before. Then we incubated them with DAPI (Servicebio, Wuhan, China) for 10 min at room temperature and washed them 3 times again. Finally, after the oocytes were mounted on glass slides, a DM3000 LED microscope (Leica, Germany) was used to observe.

Chromosome spread

Oocytes were exposed to Tyrode’s buffer (Sigma, St. Louis, MO, USA) to remove the zone pellucida. Then they were transferred to the M2 medium (Sigma, St. Louis, MO, USA) once observing the disappearance of zone pellucida. After being washed in M2 medium for 5 min, about 20 oocytes were ruptured in 20 µl spreading solution on the cover slide. They were dried completely in a ventilated place, washed three times by PBST, used DAPI (Servicebio, Wuhan, China) to dye chromosomes, and covered with coverslips. Finally, the number of spread chromosomes could be counted under the DM3000 LED microscope (Leica, Germany). The spreading solution includes 1% paraformaldehyde (Sigma, St. Louis, MO, USA), 0.15% Triton X-100 (pH = 9.2) (Sigma, St. Louis, MO, USA), and 3 mM Dithiothreitol (DTT).

IVF and embryo culture

A 10-week-old male ICR mouse was sacrificed to obtain sperm and incubated for 1 h for capacitation in the human tubal fluid (HTF) medium (MR-070, Merck Millipore). The sperm density was observed by absorbing 1 µl semen from the droplet edge, diluting 100 times with PBS, and measuring concentration with a blood cell counting plate. Then dispersed sperm and the MII oocytes were added to 50 µl HTF (Merck Millipore). The amount of semen added was calculated by the sperm density measured before to ensure the sperm was at 1 × 106/ mL. After 4 to 6 h of oocyte-sperm coincubation, zygotes were washed with the pipette. Fertilized oocytes were transferred into KSOM (MR-106-D, Merck Millipore) and cultured until the blastocyst stage. The two-cell embryos and blastocyst formation rate were calculated at 1 and 4 days after fertilization respectively.

Single oocyte RNA sequencing and analysis

After IVM with (control groups, n = 3) or without (AA groups, n = 3) AA for 6 h, COCs were digested by hyaluronidase (H3506, Sigma, St. Louis, MO, USA) to obtain oocytes. A Discover-sc™ WTA Kit V2 (N711, Vazyme, Jiangsu, China) was used to reverse-transcribe total oocyte RNA into cDNA according to the manufacturer’s instructions. A TruePrep™ DNA Library Prep Kit V2 for Illumina (TD503, Vazyme, Jiangsu, China) was used to construct the libraries. Library sequencing and analysis were performed by Illumina HiSeq X platform (Shanghai, China). The RNA-seq data were analyzed to observe the whole clustering profile by the psych package in R. The PCA selected highly variable genes (coefficient of variation > 1) and the PCA plot was mapped using the ggplot2 package in R studio. DEGs were identified using a DESeq2 package. GO enrichment analysis was performed with the database for Annotation, Visualization, and Integrated Discovery (DAVID).

Analysis of the mitochondrial membrane potential (MMP)

KGN cells were incubated in DMEM/F12 medium (Sigma, St. Louis, MO, USA) with JC-1 assay kit (Invitrogen, Carlsbad, CA) in the dark at 37 °C for 30 min, followed by 3 washes with PBS. The fluorescence intensities of green fluorescent J-monomers and red fluorescent J-aggregates were captured by fluorescence microscopy (Leica Germany). The fluorescence intensities of KGN cells were estimated by ImageJ software (NIH, Bethesda, MD, USA).

JASPAR bioinformatic analysis

JASPAR (http://jaspar.genereg.net/) database was used to predict and generate a visual analysis of the transcription factor (TF) binding to AREG promoter region.

Luciferase reporter assay

Based on the mouse AREG mRNA sequences in GenBank, the promoter of AREG was amplified and cloned into a pGL3-promoter luciferase reporter vector. KGN cells were co-transfected with AREG-promoter or/and pCMV-flag-FoxO1 vectors, together with luciferase plasmids. After 24 h, the cells were lysed using RIPA buffer. The Dual-Glo dual luciferase reporter assay system (Promega, Beijing, China) was utilized here to analyze and calculate the ratio of luminescence intensity.

Statistical analysis

All analyses were performed using GraphPad Prism 9.0 statistical software (San Diego, CA, USA) and statistical comparisons were analyzed by Student’s t test (*P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Data are presented as the mean ± SEM. P < 0.05 was considered statistically significant.