All chemicals and culture media were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise.
Animal Care and Ethics Statement
CD1 mice were used in this study. The protocols were approved by the Animal Care and Use Committee of Guangdong Medical University and were performed according to the Institutional Animal Care and Use Committee of Guangdong Medical University, People's Republic of China. 6-8-week-old female CD1 mice were used in this study. The study in humans was approved by the Committee of Medical Ethics. All the patients provided written informed consent. GV oocyte collection was conducted at the Hospital of Huizhou Reproductive Medicine Center.
GV oocytes collection and culture
CD1 mice were used for oocyte collection. To collect fully grown GV oocytes, mice were super ovulated with 5 IU of pregnant mare serum gonadotropin via intraperitoneal injection. For in vitro maturation, oocytes were cultured in M2 medium for approximately 14 h under liquid paraffin oil at 37°C in a 5% CO2 atmosphere.
In humans, GV oocytes are not of clinical use and are typically discarded according to the policy of in vitro fertilization (IVF) program. The discarded immature oocytes were only considered for research if patients had signed an informed consent form for the use of their discarded materials. The study used immature oocytes that had been aspirated during oocyte retrieval from patients undergoing IVF/ intracytoplasmic sperm injection between May 2019 and May 2022 in our IVF program. Seventy-eight human oocytes were collected over two years (< 35 years). Human GV oocytes were frozen (VT101) and unfrozen (VT102), according to the manufacturer’s instructions. The freezing process was carried out by doctors at the Reproduction Medicine Center of Hui Zhou Municipal Central Hospital, stored in a liquid nitrogen tank, and transported to the laboratory of the School of Life Sciences, Sun Yat-sen University. Prior to the experiment, an un-freezing operation was performed. Further, the immature oocytes were cultured in vitro in G-IVF PLUS medium for 24–30 h under liquid paraffin oil at 37°C in a 5% CO2 atmosphere.
The statistical standard for mouse and human oocyte maturation rate is to blow and suck the oocytes under a microscope to prevent misjudgment due to the spherical shape of the oocyte, morphological assessment, and polar body extrusion, which is a sign of oocyte maturation.
BaP and NA treatment
BaP powder was purchased from Sigma and prepared as 5 mM stock solubilized in dimethyl sulfoxide (DMSO). The stock was stored at -20°C, freshly diluted appropriately in culture medium to obtain a final concentration with equivalent vehicle DMSO (0.1%) as negative control. Women exposed to mainstream smoke have approximately 5 nM BaP in their follicular fluid [20]. Thus, 5 nM and a relatively high concentration of 50 nM were used.
NA powder was purchased from Sigma (N0761) and added to the M2 maturation medium for in vitro supplementation. According to a previously reported concentration of oocytes treated with NA of 25–50 µM [54], we conducted NA concentration gradient screening to a final concentration of 7.5, 15, and 60 µM.
In the mouse studies, BaP dissolved in DMSO was diluted in M2 medium to produce final concentrations of 5 nM and 50 nM. The negative control group was treated with the same amount of DMSO (CTRL group). NA was dissolved in 5 nM or 50 nM of BaP-contaminated M2 medium to yield final concentrations of 7.5, 15, and 60 µM.
In humans, we explored the toxic effects of 5 nM of BaP on GV oocyte maturation. BaP dissolved in DMSO was diluted in the G-IVF PLUS medium to produce a final concentration of 5 nM. The control group was treated with the same amount of DMSO (CTRL group). NA was dissolved in 5 nM of BaP-contaminated culture medium to obtain a final concentration of 15 µM.
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 [55, 56]. Commonly, oocytes prepared in advance were separated by 15% SDS-PAGE gel, transferred to PVDF membrane, blocked in 5% low-fat dry milk in one hour, then incubated with antibodies overnight at 4°C (rabbit anti-Sirt1 antibody, 1:1000). Secondary antibodies for 1 hour at 37°C after several washes, the protein bands were detected by Bio-Rad Chemi Doc XRS+. Membranes were washed with a stripping buffer and incubated with an anti-actin antibody (mouse anti-Actin antibody 1:3000) as a control.
Immunofluorescence
The specific operations were based on our pre-published protocol [55, 56]. Samples were mounted on anti-fade medium and examined under a Laser Scanning Confocal Microscope. Oocytes were fixed with 4% paraformaldehyde for 30 min, incubated with 0.5% Triton X-100 for 20 min, and 1% bovine serum albumin (diluted with phosphate-buffered saline) for 1 h. Samples were incubated with primary antibodies overnight at 4°C and with a secondary antibody for 1 h at 25°C.
For F-actin staining, MII oocytes were fixed in 3.7% paraformaldehyde for 5 min and blocked in 1% bovine serum albumin for 1 h. The samples were incubated with FITC-conjugated phalloidin for 1 h. DNA was counterstained with propidium iodide (PI) or Hoechst 33342 for 10 min. The samples were mounted on an anti-fade medium and examined under a laser scanning confocal microscope (Leica).
The fluorescence intensity was measured using ImageJ software (v1.8.0). The average fluorescence intensity of the images was determined; however, it is important to note that immunofluorescence is a semi-quantitative analysis. After ImageJ opens the image, a single channel is extracted (Image-Color-Split Channels), and the threshold is adjusted to select the appropriate region (Image-Adjust-Threshold). If none of the algorithms of the threshold select the area of the signal well, we can circle the area with the signal. For example, the fluorescence intensity of the cell inside or in the membrane is measured. We set the parameters to be measured (Analyze-Set Measurements) and perform the detection (Analyze-Measure).
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. The DNA was counterstained with Hoechst 33342 for 10 min. The oocytes were then moved to a live cell-imaging dish after washing. Fluorescence was observed using a confocal microscope (Leica).
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. The fluorescence intensity was measured using ImageJ software.
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. The fluorescence intensity was measured using ImageJ software.
Statistical analysis
Data were showed as mean ± SEM. Statistical comparisons were used Student’s t test and the multiple comparisons between more than two groups were analyzed by ANOVA Tukey's multiple comparison test. The analyzed were performed using GraphPad Prism 8 software. p < 0.05 was considered significant.