All chemicals and culture media were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise.
Animal Care And Ethics Statement
In mouse part, CD1 mice were used in this study. Protocols were approved by the Animal Care and Use Committee of Sun-Yat-Sen University, and performed according to the institutional Animal Care and Use Committee of Sun Yat-Sen University, People's Republic of China. CD1 mice were used 6-8week-old female mice in this study.
In human part, the study was approved by the Committee of Medical Ethics. All patients in this study had written their informed consent. Germinal vesicle (GV) oocytes collection was conducted at the Hospital of Huizhou Reproductive Medicine Center.
Gv Oocytes Collection And Culture
In mouse part, CD1 mice were used for oocyte collection in this study. To collect fully grown germinal vesicle (GV) oocytes, mice were super ovulated with 5 IU Pregnant Mares Serum Gonadotropin (PMSG) by intraperitoneal injection. For in vitro maturation, oocytes were cultured in M2 medium about 14 hours under liquid paraffin oil at 37°C in a 5% CO2 atmosphere.
In human part, GV oocytes were clinically useless, these are typically discarded according to the policy of IVF program. The discarded immature oocytes were only released for this research if patients had signed an informed consent for use of their discarded materials. The study used immature oocytes that had been aspirated during oocyte retrieval from patients undergoing IVF/ICSI patients between May 2019 and May 2022 in our IVF program. 78 human oocytes were collected over 2 years (༜35 years). The human GV oocytes were frozen (VT101) and unfrozen (VT102) according to the instructions of the reagents. The freezing process was carried out by doctors in the Reproduction Medicine Center of Hui Zhou Municipal Central Hospital, then stored in a liquid nitrogen tank, and transported to the laboratory of the School of Life Sciences, Sun Yat-sen University. Before the experiment, the unfrozen operation was performed. Then, the immature oocytes were cultured in vitro in G-IVF PLUS medium about 24–30 hours under liquid paraffin oil at 37°C in a 5% CO2 atmosphere.
Bap And Na Treatment
In mouse part, BaP dissolved in DMSO was diluted in M2 medium to produce a final concentration of 5nM and 50nM respectively. The negative control group added the same amount of DMSO, called CTRL group. NA was dissolved in 5nM or 50nM BaP contaminated culture medium to yield a final concentration of 7.5µM,15µM,60µM.
In human immature part, BaP dissolved in DMSO was diluted in G-IVF PLUS medium to produce a final concentration of 5nM. The control group added the same amount of DMSO. NA was dissolved in 5nM BaP contaminated culture medium to yield a final concentration of 15µM.
Antibodies
Rabbit polyclonal anti-Sirt1 antibody was purchased from Abcam (Cat#: ab32441); Rabbit polyclonal anti-gamma H2A.X (phosphor-S139) antibody was purchased from Abcam (Cat#: ab2893).
Western Blotting
100 mouse oocytes were lysed in laemmli sample buffer with protease inhibitor and boiled for 5 minutes. The specific experimental operations were based on our pre-published protocol (51, 52).
Immunofluorescence
The specific operations were based on our pre-published protocol (51, 52). Samples were mounted on anti-fade medium and examined under a Laser Scanning Confocal Microscope.
Mitochondria Distribution Measurement
Mito Tracker was labeled the mitochondria. Living oocytes were cultured in M2 medium containing 200 nM Mito Tracker Red (Molecular Probes, Eugene, OR) for 30 min at 37°C.
Mitochondrial Membrane Potential Measurement
Oocytes were cultured in M2 medium containing 2µΜ JC-1 (Thermo Fisher Scientific, Cat#: T3168) for 30 min at 37°C. The chromosomes were counterstained with propidium iodide (PI) or Hoechst33342 for 10 minutes. Oocytes were transferred to a live cell-imaging dish after 3 washes. Fluorescence was detected by a Leica inverted microscope.
Ros Measurement
Living oocytes were incubated in M2 medium with 5µΜ CM-H2DCFDA (Life Technologies, Invitrogen TM, Cat#:C6827) for 30 min at 37°C in 5% CO2 atmosphere. Oocytes were transferred to a live cell-imaging dish after 3 washes, and observed using a Confocal Microscope.
Statistical analysis
Data were showed as mean ± SEM. Statistical comparisons were used Student’s t test and analyzed were performed using GraphPad Prism software.