3 isobutyl 1 methyl xanthine (ibmx)

For the animal experiment, ICR mice aged 40~48 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Totally, 18 aged female mice were sacrificed by cervical dislocation and 241 oocytes were obtained. All the experiments were repeated three independent times. Specifically, the bilateral ovaries were isolated, transferred into M2 medium (EasyCheck, Nanjing, China) containing IBMX (Selleck, Houston, TX, USA), and punctured with sterile needles to release immature oocytes. After being washed five times in M2 medium, the denuded GV oocytes were moved to the IVM medium (EasyCheck, Nanjing, China), with or without 10 nM rapamycin (MedChemExpress, Monmouth Junction, NJ, USA), and then cultured for 16 h at 37°C under 5% CO₂ in humidified air. Following IVM, MII oocytes were recognized with the extrusion of the first polar body (PB1), and the maturation rates of the control group and rapamycin group were recorded and compared.

Celecoxib (Cat# PHR1683), safranin O solution (Cat# HT90432), fast green (Cat# F7252), hematoxylin solution (Cat# MHS1), eosin Y solution (Cat# 318906), dimethyl sulfoxide (DMSO, Cat# D8418), sodium citrate (Cat# PHR1416), hydrogen peroxide (Cat# 18304), Triton X-100 (Cat# T8787) and TRAP staining kits (Cat# 387 A) were purchased from Sigma-Aldrich (St. Louis, MO). Grapiprant (Cat# S6694), PF-04418948 (Cat# S7211), AZD6244 (Cat# S1008), JNK-IN-8 (Cat# S4901), SB203580 (Cat# S1076), GSK2141795 (Cat# S7492), neferine (Cat# S5144), IBMX (Cat# S5836) and PGE2 (Cat# S3003) were purchased from Selleck (Houston, TX). Recombinant mouse M-CSF protein (Cat# 416-ML) and recombinant mouse RANK L protein (Cat# 462-TRL) were purchased from R&D (Minnesota, USA). Matrigel (Cat# 356234) was purchased from BD Biosciences (San Jose, CA). Mountant (Cat# P36931) was purchased from Thermo Fisher Scientific (Waltham, MA). A TRACP staining kit (Cat# MK300) was purchased from TaKaRa (Dalian, China).

Drugs used in the paper include ERK inhibitor
Vx-11e (Selleck Chemicals), SCH772984 (Selleck Chemicals), KO-947
(MedChem Express), Ulixertinib (Selleck Chemicals), and LY3214996
(Selleck Chemicals); MEK inhibitors U0126 (Tocris), PD0325901 (Selleck
Chemicals, ApexBio), and GSK1120212 (Selleck Chemicals; ApexBio),
JNK inhibitor SP600125 (Selleck Chemicals), p38 inhibitor PD169316
(ApexBio), Forskolin (Selleck Chemicals), and IBMX (Selleck Chemicals).
Antibiotics used in cell culture include blasticidin (Invivogen),
geneticin (Invitrogen), penicillin/streptomycin (Gemini Bio), ciprofloxacin
(Sigma-Aldrich), and piperacillin (Sigma-Aldrich).