Animals and drug administration. Six-week-old and 8-month-old female C57BL/6 mice and 3-week-old female ICR mice were purchased from SPF Biotechnology Co., Ltd (Beijing, China). The mice were raised until experimental time in the Animal Laboratory Center of Nanjing Drum Tower Hospital under a 12-h light/dark cycle at a constant temperature (20–23°C) and had free access to food and water according to the institutional guidelines. Nine-month-old female C57BL/6 mice were intraperitoneally injected with 5 mg kg− 1 MVA (Sigma, St. Louis, MO, USA; M4667) every day at 18:00 hours for 30 consecutive days. 9.5-month-old female C57BL/6 mice were intragastrically gavaged with 5 mg kg− 1 8-IPF (Yuanye, Shanghai, China; B21576) every day at 18:00 hours for 14 consecutive days. Approval for the procedures involving mouse protocols and experiments was obtained from the Experimental Animal and Welfare Ethics Committee of Nanjing Drum Tower Hospital (2023AE01053).
COC and DO IVM in MEMα maturation medium. Female mice were intraperitoneally injected with 10 IU pregnant mare serum gonadotropin (PMSG) (Sansheng Pharmaceuticals, Ningbo, China) and sacrificed 48 h later. COCs and DOs were isolated from the ovarian antral follicles using a disposable syringe with a 20-gauge needle and subsequently cultured in MEMα (Gibco, Waltham, MA, USA; 32561037) maturation medium covered with liquid paraffin oil in an incubator at 37 ℃ with 5% CO2. The MEMα maturation medium contained 10% foetal bovine serum (FBS) (Gibco, Waltham, MA, USA; 10270106), 10 ng/mL epidermal growth factor (EGF) (Gibco, Waltham, MA, USA; 53003-018), and 1.5 IU/mL human chorionic gonadotropin (hCG) (Sansheng Pharmaceutical, Ningbo, China). After culturing in IVM for 4 h and 14 h, respectively, the GVBD and PBE rate were analysed. 50 µM MVA, 10 µM FOH (Sigma, St. Louis, MO, USA; F203), and 10 µM FTI-277 (Aladdin, China; F331609) were administered by addition to the MEMα maturation medium in this study.
Oocyte immunofluorescence. Oocytes were fixed in 4% paraformaldehyde (PFA) (Sigma, St. Louis, MO, USA; 158127) for 30 min before permeabilizing in 0.5% Triton X-100 (Sigma, St. Louis, MO, USA; T9284) for 20 min. Then, blocking was performed using 1% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA; 10711454001) for 1 h. Oocytes were incubated with mouse monoclonal anti-α-tubulin-FITC (Sigma, St. Louis, MO, USA; F2168; 1:200), 594-phalloidin (US Everbright® Inc., Suzhou, China; YP0052L, 1:200), mouse monoclonal anti-Arp3 (Santa Cruz Biotechnology, California, USA; sc-48344, 1:100), mouse monoclonal anti-CDC42 antibodies (Santa Cruz Biotechnology, California, USA; sc-8401; 1:200) and mouse monoclonal anti-RAC1 antibodies (Santa Cruz Biotechnology, California, USA; sc-514583; 1:200) at 4°C overnight. The oocytes were then treated with a secondary antibody for 1 h at room temperature. After three 5 min washes in phosphate-buffered saline with 0.05% Tween 20 (PBST), the oocytes were incubated with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA; H3570) for 10 min at room temperature. Then, the oocytes were mounted on glass slides and observed under a fluorescence microscope (Leica, Germany; DM3000).
Chromosome spread. MII oocytes were first treated with Tyrode’s buffer (Sigma, St. Louis, MO, USA; T2397) for 3 min at 37°C to remove the zona pellucida. Then, the cells were cultured in M2 medium (Sigma, St. Louis, MO, USA; M7167) for 10 min and fixed in 1% PFA with 0.15% Triton X-100 (pH 9.2) on a glass slide. After drying, the samples were incubated with a human anti-centromere antibody (Incorporated, Davis, CA, USA; CA95617; 1:100) at 4°C overnight. After three 5 min washes in PBST, the samples were incubated with Hoechst 33342 for 10 min at room temperature. Finally, the number of spread chromosomes was counted under a DM3000 LED microscope (Leica, Germany).
In vitro fertilization (IVF) and embryo culture. The sperm from the epididymides of 12-week-old C57BL/6 male mice were collected and capacitated for 1 h in human tubal fluid (HTF) medium (Merck Millipore, St. Louis, MO, USA; MR-070). COCs after IVM for 14 h or obtained from oviductal ampullae of C57BL/6 female mice were added to be fertilized by the addition of capacitated sperm for 6 h in a 37°C incubator with 5% CO2. The fertilized oocytes were subsequently transferred to KSOM (Merck Millipore, St. Louis, MO, USA; MR-106-D) for subsequent culture. The 2-cell embryos and blastocysts formation rates were subsequently calculated.
Metabolite extraction and targeted metabolomics analysis. Metabolites were extracted using a method described previously with some modifications48. Mice oocytes and GCs were briefly washed with PBS after isolation before quenching. To quench cells and stop metabolism, oocytes and GCs were harvested in tubes and quick-frozen in liquid nitrogen immediately after washing. Oocytes or cells were then incubated in an extraction buffer containing 80% MS grade methanol and 2 µg/ml 4-CL-phenylalanine (Sigma, St. Louis, MO, USA; C6506) as an internal standard which was precooled at -80°C freezer. Metabolites were extracted by 3 rounds of bead-beating. After two rounds of centrifugation by 15,000 g for 10 minutes at 4°C, the supernatant fraction containing soluble metabolites was harvested and dried using a CentriVap Concentrator system (Labconco).
For running samples, a dried metabolite extract sample was re-suspended in 50% MS grade acetonitrile for injection. MVA pathway metabolites such as FPP and GGPP levels were quantitatively analyzed in a negative ion mode using multiple reaction monitoring (MRM) acquisition with a triple quadrupole mass spectrometer (Triple Quad 6500+, AB SCIEX) that is coupled to a high performance liquid chromatography. A mix standard containing FPP (Sigma, St. Louis, MO, USA; F6892) and GGPP (Sigma, St. Louis, MO, USA; G6025) with series dilution for 20 ng/ml, 40 ng/ml, 200 ng/ml, 500 ng/ml, 2 µg/ml, 5 µg/ml was used to make standard curves. Metabolites were separated chromatographically on a UPLC HSS T3 column (ACQUITY 1.8 µm 150 x 2.1 mm, Waters). Flow rate was set to 0.25 mL/min using the following method: Buffer A: 10 mM ammonium carbonate, Buffer B: 100% acetonitrile. T = 0 min, 10% B; T = 1 min, 10% B, T = 4 min, 65% B; T = 6 min, 65% B; T = 6.5 min, 95% B; T = 8.5 min, 95% B; T = 9 min, 10% B; T = 12 min, 10% B stop. The retention time for each MRM peak was compared to an appropriate standard. The area under each peak was then quantitated by using SCIEX OS software and re-inspected for accuracy.
RNA-seq library construction and analysis. Oocytes and GCs isolated from COCs were lysed to obtain cDNA using a Discover-sc WTA Kit V2 (Vazyme Biotech, China; N712). A TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech, China; TD503) was used for library construction. The Illumina HiSeq X platform (Nanjing Genemap Co., Ltd., China) was used to perform sequencing. High-quality reads were aligned to the Mus musculus UCSC mm9 reference genome, and the FPKM value of each gene was calculated. Highly variable genes (coefficient of variation > 1) were selected for PCA, and the resulting PCA plot was generated using the ggplot2 package in R studio. The DESeq2 package was used to identify DEGs. KEGG enrichment analysis was performed with KEGG Mapper.
qRT‒PCR. All primers were mixed in nuclease-free water to a final concentration of 0.1 µM as a primer assay pool. For each mixture, 5 µL of solution was prepared as follows: 2.5 µL of reaction mixture, 0.5 µL of primer mixture, 0.1 µL of RT-Taq mixture, and 1.9 µL of nuclease-free water (Vazyme Biotech, China; P621). GCs isolated from COCs were added to the mixture and placed in a -80°C freezer for 2 min. The mixture was centrifuged at 1000 g for 2 min, after which PCR was performed. For qRT‒PCR, 5 µL of SYBR-Green (Vazyme Biotech, China; Q121), 0.5 µL of Primer-forward (10 µM), 0.5 µL of Primer-reverse (10 µM), 2 µL of cDNA, and 2 µL of ddH2O were mixed. The following primer sequences were used: MVK (mouse): forward, 5’-AGCGTCAATTTACCCAACATCG-3’, reverse, 5’-GAGACATCACCTTGCTCAAGAAA-3’; FDPS (mouse): forward, 5’-GGGTTTGACCGTGGTACAAG-3’, reverse, 5’-AAGCCTGGAGCAGTTCTACAC-3’; GGPPS (mouse): forward, 5’-TTTTGCATACACTCGACACACT-3’, reverse, 5’-GGCCTCAATTTGTTTGTAGGCT-3’; SQLE (mouse): forward, 5’-GACCTCGTTCGTGACGGAC-3’, reverse, 5’-CTCCCCAACTATCCTGTCGG-3’; and 18S (mouse): forward, 5’-ATGGCCGTTCTTAGTTGGTG-3’, reverse, 5’-CGGACATCTAAGGGCATCAC-3’. All data were normalized to the expression of 18S using the comparative 2−ΔΔCt method.
Cell culture and metabolic labelling. Human KGN cells were cultured in DMEM/F12 (Gibco, Waltham, MA, USA; C11330500BT) supplemented with 10% FBS (Gibco) and 1% penicillin‒streptomycin (Gibco, Waltham, MA, USA; 15140122) in a humidified 37°C incubator with 5% CO2. Cells were grown in 10 cm plates to approximately 70% confluence and treated with alk-FOH (20, 50, or 100 µM; from 100 mM stock solution in DMSO) or DMSO only for 48 h. For antagonistic coincubation, both 50 µM alk-FOH and 50 µM FOH (Sigma, St. Louis, MO, USA; F203) were added to KGN cells for 48 h. Then, the cells were washed three times with cold phosphate-buffered saline. The cells were lysed for western blotting and pull-down assays.
Western blotting. The proteins were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA; 23227) following the manufacturer’s instructions. Next, the equivalent proteins were separated via 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, St. Louis, MO, USA; 03010040001). The membranes were incubated with primary antibodies, rabbit polyclonal anti-Farnesyl (Invitrogen, Waltham, MA, USA; PA1-12554; 1:1500), FDPS (Abcam, MA, USA; ab153805; 1:1000), MVK (Proteintech, China; 12228-1-AP; 1:1000), rabbit monoclonal anti-CDC42 (Abcam, MA, USA; ab187643; 1:1000), rabbit monoclonal anti-RAC1 (Cell signaling Technology, Danvers, MA, USA; 4651S; 1:1000), rabbit monoclonal anti-Arp2 (Abcam, MA, USA; ab128934; 1:1000), rabbit monoclonal anti-Arp3 (Abcam, MA, USA; ab181164; 1:1000), rabbit monoclonal anti-N-WASP (Abclonal, Wuhan, China; A2270; 1:1500), mouse monoclonal anti-WAVE2 (Abclonal, Wuhan, China; A19601; 1:1500), rabbit monoclonal anti-β-actin (Bioworld, Beijing, China; AP0060; 1:100000), and rabbit polyclonal anti-Calnexin (Proteintech, China; 10427-2-AP; 1:10000) at 4℃ overnight after blocking with 5% nonfat milk in PBST for 1 h at room temperature. The next day, the PVDF membranes were washed three times with PBST, incubated with HRP-conjugated goat anti-rabbit IgG (Abcam, MA, USA; ab97051; 1:10000) at room temperature for 1 h, washed three times with PBST, and then developed using enhanced chemiluminescence reagents (GE, Piscataway, NJ, USA); the mean grey value was estimated with ImageJ software (NIH, USA, Version 1.0).
Cu-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction. Metabolic labelling and protein extraction were performed as described above. Protein concentrations were adjusted to 2.2 mg/mL. For each, 3000 µL of protein lysate click reagent mixture was prepared as follows: 2520 µL of protein (2.2 mg/mL), 60 µL of azide biotin (Confluore, China; 908007-17-0) (0.1 mM, 5 mM stock solution in DMSO), 120 µL of BTTAA-CuSO4 (2:1, 1 mM:0.5 mM), and 300 µL of fresh sodium ascorbate (2.5 mM, 25 mM stock solution in PBS), which were added last. Then, the reaction mixture was incubated on a shaker at room temperature for 3 h. After the CuAAC reaction, the metabolic labeled proteins were precipitated overnight in methanol at -40°C. The next day, the samples were centrifuged at 5,000 × g for 15 min at 4°C and subsequently washed twice with 7.5 mL of precooled methanol. The precipitated proteins were subsequently completely dissolved in 1.67 mL of buffer (1.2% SDS in PBS) after the methanol was discarded, after which the mixture was left at room temperature for 20 min of volatilization. One hundred microlitres of Pierce High Capacity Streptavidin Agarose (Thermo Fisher Scientific, Waltham, MA, USA; 20359) was washed with PBS three times and resuspended in 8.33 mL of PBS in a 15 mL tube. The redissolved proteins were added to 15 mL tubes filled with the agarose and incubated gently with rotation at room temperature for 4 h. The nonspecific binding proteins were then washed with 0.2% SDS in PBS (10 mL, once), PBS (10 mL, three times), and ddH2O (10 mL, three times) and centrifuged at 500 g for 1 min, after which the beads were transferred to 1.5 mL centrifuge tubes with ddH2O. Then, 2× loading buffer (Beyotime, Shanghai, China; P0015L) was added to the precipitated beads, which were subsequently heated at 95°C for 12 min. Next, the samples were centrifuged, after which approximately 40 µL of 2× loading buffer was transferred to a new 1.5 mL tube to obtain the protein released from the beads. The proteins were subsequently separated via 10% SDS-PAGE for Western blotting or were subjected to LC‒MS/MS.
LC‒MS/MS analysis. The enriched protein samples were analysed using an Easy-nLC 1000 (Thermo Fisher) (Buffer A: 0.1% formic acid solution; Buffer B: 0.1% formic acid + 80% acetonitrile solution; Thermo Fisher, USA). After the column was equilibrated with 95% Buffer A, the sample was loaded onto a Trap column. Then, the samples were separated by chromatography and analysed via mass spectrometry (MS). MS analysis was performed with a Q Exactive mass spectrometer (Thermo Fisher, USA). The raw MS files were searched using MaxQuant 1.6.14 and compared to the Homo sapiens database downloaded from UniProt and finally the identified protein results were obtained.
CuAAC reaction for fluorescence imaging. COCs were incubated with 50 µM alk-FOH for 9 h. COCs were fixed with 4% PFA for 30 min. Then, the COCs were incubated with 50 µM azide AZDye 488, the BTTAA-CuSO4 complex (BTTAA/CuSO4 6:1), and 2.5 mM sodium ascorbate in PBS at room temperature for 30 min. After the click reaction, COCs were incubated with 594-Phalloidin and Hochest 333342 at room temperature for 30 min. Fluorescence imaging was subsequently performed under LSM 900 confocal laser scanning microscope (Zeiss).
Total, membrane and cytoplasmic protein isolation. Total proteins were isolated by lysing cells with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China; P0013B) supplemented with protease inhibitors at 4°C for 30 min. The supernatant of the lysates was collected after centrifugation at 12,000 rpm for 15 min.
Cell membrane and cytoplasmic proteins were isolated using a Membrane and Cytosol Protein Extraction Kit with protease inhibitors according to the manufacturer’s instructions (Beyotime, Shanghai, China; P0033). In brief, approximately 3 × 107 cells were homogenized using 1 mL of membrane protein extraction reagent A supplemented with PMSF and protease inhibitors for 30 min at 4°C. The nuclei and unbroken cells were removed by centrifuging at 700 g for 10 min. The supernatant was then collected for further centrifugation at 12,000 rpm for 30 min to obtain the plasma proteins. The remaining sediment was fully vortexed in 200 µl of membrane protein extraction reagent B on ice for 10 min and subsequently centrifuged at 12,000 rpm for 30 min to obtain the membrane proteins.
Coi
mmunoprecipitation (co-IP) assay. KGN cells were treated with 10 µM FOH for 48 h, total protein was extracted with RIPA buffer supplemented with protease inhibitor, and total protein was quantified with a BCA protein assay kit. Next, 500 g of total protein was incubated with 1 g of anti-mouse immunoglobulin G (IgG), anti-CDC42 (Santa Cruz Biotechnology, California, USA; sc-8401), or anti-RAC1 (Santa Cruz Biotechnology, California, USA; sc-514583) at 4°C overnight. Then, 30 L of agarose beads was added to the cell lysates, which were incubated at 4°C for 4 h. Subsequently, the cell lysates were incubated with 2 × loading buffer at 95°C for 10 min. Finally, Western blotting was performed to analyse the protein samples.
Expression Constructs, mRNA Synthesis and microinjection. The mcherry coding sequences and CDC42 or RAC1 coding sequences were fused and inserted into pGEMHE plasmids to obtain pGMHE-mCherry-CDC42 and pGMHE-mCherry-RAC1. pGMHE-mCherry-CDC42C188Y(M-CDC42) and pGMHE-mCherry-RAC1C188Y(M-RAC1) were obtained using a site-directed mutagenesis kit (NEB, E0554) for in vitro transcription. After linearization of the template with AscI (NEB, R0558V), capped mRNA was synthesized using HiScribe T7 High yield RNA Synthesis Kit (NEB, E2040S), and dephosphorylated for uncapped RNA using Antarctic phosphatase (NEB,M0289). Finally, purified for phenol/chloroform and dissolve in 11 µl nuclease-free H2O for mRNA. mRNA concentrations were determined by NanoDrop (Thermo Fisher Scientific).
Mouse oocytes were microinjected with 3.5 pl of mRNA. mRNA was microinjected at a needle concentration (final concentration in the microinjection needle) of 100 ng/µl. Oocytes were allowed to express the mRNAs for 3 h before release in M2 medium containing 250 µM dbcAMP (Sigma-Aldrich). After 3 h, the oocytes were maintained in dbcAMP-free M2 medium and performed using LSM 900 confocal laser scanning microscope (Zeiss).
Body weight and ovarian index. The mice in each group were euthanized after weighing. A U-shaped incision in the hypogastrium was made to explore the main organs. The bilateral ovaries connected to the bicorned uterus via the fallopian tubes were separated, and the peri-ovarian adipose tissue was removed under a stereoscope (Leica, Germany). The ovaries were subsequently weighed on an electronic analytical balance when no liquid remained. A representative picture was taken on dry sterile gauze, and the ovarian index was determined based on the ovarian wet weight (mg) / body weight (g).
Histological analysis and follicle counting. The oestrous cycles of the mice were detected by daily examination of vaginal smears, and the mice were sacrificed in the dioestrus phase to collect ovaries. Ovaries were fixed with 4% paraformaldehyde in PBS overnight, dehydrated in 70%, 80%, 90%, 95%, and 100% ethanol, cleared with xylene and embedded in paraffin. The ovarian tissues were serially sectioned at 5 µm, sequentially deparaffinized in xylene, rehydrated in a descending series of graded ethanol solutions, and stained with haematoxylin and eosin (HE).
One of every five slices was used to count the follicles. The follicles at the primordial, primary, secondary, and antral stages in the histological sections of the ovaries were observed and classified based on the histological morphology under a microscope at 200× magnification. Only follicles containing a visible nucleus were counted independently by two researchers. The result was calculated as a five-fold counting value.
Fertility test. After two weeks of gavage, to evaluate the fertility function after 8-IPF administration, female mice were mated with 10-week-old C57BL/6 male mice with proven fertility. At 8:00 AM the next day, vaginal plugs were observed to confirm whether the female mice were pregnant. Female mice were considered infertile if, after continuous mating for two-weeks, no vaginal plugs were observed. The pregnancy was terminated by caesarean section at 18.5 days gestation, and after routine anaesthesia, the uterus was exposed through a U-shaped incision in the lower abdomen. The pregnancy and embryo implanted conditions of the mice were observed and photographed, after which the embryos and placenta were separated, weighed and recorded. The number of viable offspring in each litter was recorded.
Statistical analysis. Normally distributed data are presented as the mean ± standard error of the mean (SEM) and were compared by unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Continuous variables without a normal distribution are presented as medians and interquartile ranges and were analysed by the Mann‒Whitney U test. All the statistical comparisons were performed with GraphPad Prism 8.0.
Schematics. Schematic cartoons in Fig. 1a, 2a, 3a, 4a, 5g, 6a, 7d and Extended Data Fig. 6c, 7e were created with BioRender.com.