Patients
According to the study design, female patients receiving IVF treatment at the Reproductive Medicine Center of Ningxia Medical University General Hospital from January 2021 to September 2022 were collected. The diagnostic criteria for DOR patients included in the study were as follows: (1) Age ≤ 35; (2) The number of oocyted obtained is less than 5; (3) FSH > 10UI/L; (4) AMH < 1.1ng/mL. The inclusion criteria of control group: (1) age ≤ 30; (2) The number of eggs obtained is 9–15; (3) FSH < 10UI/L; (4) 2ng/mL < AMH < 6.8ng/mL. A total of 278 cases of funicular fluid in female patients during oocyte pick-up (OPU) were collected, and clinical data such as hormone values of the patients were obtained, including 129 cases of DOR and 149 cases of control group. 3 cases of funicular fluid in each group were used for genome sequencing, 10 cases in each group for sequencing results verification, and the remaining patients were used for correlation analysis.
This study was approved by the Ethics Committee of General Hospital of Ningxia Medical University (Ethics review No.KYLL-2021-235).
Exosome isolation and characterization
The follicle fluid, plasma and culture medium were collected and centrifuged at 3000 g for 15 min to remove cells and cellular debris. Exosomes were isolated using the Exoquick exosome precipitation solution (System Biosciences). The morphology of exosomes was investigated via transmission electron microscopy (TEM; HITACHI, Japen), and the number and size distribution of exosomes were analyzed by nanoparticle tracking anal ysis (NTA) via the NANOSIGHT NS300 system (Malvern, UK). Western blot was utilized to evaluate the expression of exosomal markers
Animals
This study was conducted in strict accordance with the ethical standards set forth by the Animal Ethics Committee of Ningxia Medical University and all processes were carried out according to the Animal Research Guidelines.
6 weeks C57BL/6J female mice were obtained from the laboratory animal center of Ningxia Medical University.Mice were housed in a specific pathogen-free (SPF) conditions at 24 ± 1 ℃, 40–70% humidity and with a 12/12h light/dark cycle, fed and watered ad libitum. Mice were randomly divided into control and DOR groups. The DOR group received intraperitoneal injections of CDDP (2.5 mg/kg/d in saline) (Sigma-Aldrich, Shanghai, China) for 7 consecutive days, and the control group were injected with saline.
Oocyte collection and in vitro maturation
To collect MII oocytes, female mice were injected with 5 IU pregnant mare serum gonadotropin (PMSG) (Ningbo Second Hormone Company, 110254564), and 48 h later with 5 IU human chorionic gonadotropin (hCG) (Ningbo Second Hormone Company, 110251282). After 13.5 h, cumulus-oocyte complexes (COCs) were obtained from the oviducts and MII oocytes were collected by removing the cumulus cells using hyaluronidase digestion (Nanjing Aibei Biotechnology Co., Ltd., M2215).
To collect GV oocytes, female mice female mice were injected intraperitoneally with 5 IU PMSG. After 48 h, ovarian tissues were obtained, COCs were isolated from ovaries in M2 (Sigma-Aldrich, M7167) medium by using a 4.5-gauge needle, and the cumulus cells were cleared away from COCs by repeated mouth-controlled pipetting. The GV oocytes were then cultured with M16 medium (Sigma-Aldrich, M7292) under mineral oil at 37℃, 5% CO2.
In vitro fertilization and embryo culture
Caudae epididymides from 12-week-old male mice were lanced in a dish of HTF medium under mineral oil to release sperm, followed by being capacitated for 1 h (37 ℃, 5% CO2), and added to ovulated oocytes at a concentration of 4 3 105/mL sperm in 100 mL HTF for 5 h at 37℃, 5% CO2. The presence of two pronuclei was scored as successful fertilization. The embryos were cultured in a 96-well culture plate containing 150 mL KSOM under 50 mL mineral oil at 37℃ in a 5% CO2 atmosphere.
Serum hormone levels determination
ELISA was conducted according to the instruction of the mouse AMH, FSH, LH, E2 ELISA Kit (JL20476-96T, JL10239-96T, JL10432-96T, JL11790-96T, Jianglai Bio., China). 50µL of standards with different concentrations were added to each standard well. Serum 10µL and 40µL diluent were added to the sample in the air, and sample diluent 50µL was added to the blank hole. Horseradish peroxidase (HRP) labeled detection antibody was added to 100µL in each well, incubated at 37℃ for 60min, and washed the plate 5 times. Add 50µL of substrate A and B to each well and incubate at 37℃ for 15min without light. Add stopper solution 50µL to each well, measure OD value of each hole at 450nm wavelength within 15min, and draw standard curve.
Estrus cycle examination
The mice underwent daily vaginal exfoliated cell smears from 17:30 to 18:00. 20 µl of saline was gently placed on the vaginal opening and rinsed 2–3 times. The rinsing solution was then coated on slides and observed under a light microscope. Mice estrous cycle was determined by the morphology and type of exfoliated cells for 14 days of observation. Proestrus (P) is characterized by predominantly nucleated epithelial cells, while estrus (E) is characterized by predominantly keratinized and non-nucleated epithelial cells. Metestrus (M) is characterized by a mixture of nucleated epithelial cells, keratinized and non-nucleated epithelial cells, and neutrophils. Diestrus (D) is characterized by a large number of neutrophils.
Histological analysis of ovaries
Ovaries used for histological analysis were collected from each group of mice and fixed in 4%paraformaldehyde (pH 7.5) overnight at 4℃, dehydrated, and embedded in paraffin. Paraffin-embedded ovaries were sectioned at a thickness of 8 mm for hematoxylin and eosin (H&E) staining. Both ovaries from three mice of each group were used for the analysis.
Immunofluorescence of ovaries
The prepared paraffin sections of ovaries were placed in dewaxing solution for 10–15 min and anhydrous ethanol for 15 min. The washed ovarian sections were incubated with EDTA antigen retrieval buffer and repaired in a microwave oven. A histochemical pen was used to draw a circle around the tissue of the slices, and BSA was dropped and incubated for 30 min, following incubating primary antibodies overnight and secondary antibodies for 50 min. DAPI dye solution was incubated at 37°C in darkness for 10 min, and autofluorescence quench was treated for 5 min. The tablets were sealed with anti-fluorescence quench sealing agent. The dewaxed sections were incubated with hematoxylin for 5 min and eosin dye solution for 3 min, then dehydrated and sealed. Finally, the sections were photographed under a fluorescence microscope.
Western blot analysis
A total of around 100 oocytes and ovaries were collected from each group. Each protein sample was extracted using a cell lysis solution containing 1% PMSF (Cell Signaling Technologies) following the manufacturer's instructions. The protein samples underwent boiling at 100°C for 5 min and subsequently separated on a 10% SDS-PAGE gel. Then, proteins were transferred to the polyvinylidene fluoride membrane (Millipore, USA) through a transmembrane transfer. Membranes were immersed in 5% skimmed milk for 2 h at room temperature, washed briefly with TBST (TBS containing 0.1% Tween 20), and incubated with primary antibodies overnight at 4℃. Rinse the membranes 3 times with TBST for 10 min, then incubate them with secondary antibody for 1 h at room temperature and rinse them 3 times with TBST for 15 min each. Finally, the signals were detected using an enhanced chemiluminescence (ECL) kit (KeyGEN BioTECH, KGC4602-200) and quantitatively analyzed in grayscale with Image J.
RNA isolation, reverse transcription, and RT–qPCR
Total RNA was extracted from oocytes and ovaries with TRIzol reagent (Takara, Japan) according to the manufacturer’s protocol, and an equal amount of RNA from each sample was extracted with a reverse-transcription kit (R233,Vazyme, China). Real-time PCR was performed using SYBR® Green gene expression assays (R711, Vazyme, China). GAPDH and U6 were respectively used as the controls for lncRNA LIPE-AS1 and miR-330-5p, and data were collected based on the comparative cycle threshold (CT) (2−ΔΔCT) method. The primers are listed in Supplementary Table 1.
Transmission electron microscopy (TEM)
Ovary tissues were obtained from 14-week-old mice and fixed in 2.5% glutaraldehyde at 4°C overnight. Subsequently, the tissues were rinsed three times for 10 min each in PBS. Then the ovary was transferred into 1% osmic acid. After 2 h, it was washed three times with PBS for 10 min each time. The ovaries were fixed with 2% uranyl acetate for 30 min and dehydrated in ethanol gradients (50%, 70%, 90%, and 100%) for 10 min each. Transferred to a solution of 90% acetone and 90% ethanol (1:1) for 20 min, then the samples were transferred to 90% acetone at 4°C for 20 min, followed by two transfers to 100% acetone for 15 min each. Finally, they were embedded in epoxy resin. Semi-thin sections after staining with 1% methylene blue in 0.5% borax were examined using an Olympus BX60 light microscope. And ultrathin sections (50 nm), stained by lead citrate, were observed using a HITACHI H-7650 transmission electron microscope (HITACHI, Japan)
Exosome weight suspension 10µL was taken and added to the sample carrying copper network. After precipitation at room temperature for 2-3min, the floating liquid was absorbed on the edge of the copper mesh with filter paper, and then negatively stained with equal volume 3% phosphotungstic acid solution for 4 min, and then absorbed with filter paper. Dry at room temperature for 3 minutes
Immunofluorescence and confocal microscopy
Oocytes were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 30 min and permeablilzed in 0.5% Triton-X-100 for 20 min at room temperature. Then, oocytes were block with 1% BSA-supplemented PBS for 1h and incubated with anti-α-tubulin-FITC antibody (Sigma-Aldrich, F2168), anti-centromere antibody (Antibodies Incorporated, ABI-15-234), anti-ovastacin antibody (Bio-Techne, AF8549) at 4℃ overnight. After washing in PBST, oocytes were incubate with fluorescently labeled secondary antibodies (according to the corresponding primary resistance).Then oocytes were counter-stained with Hoechst for 10 min. Finally, oocytes were mounted on glass slides and observed under a laser scanning confocal microscope (Nikon A1).
For active mitochondrion staining, oocytes were cultured in M16 medium containing 100 nM cell per-meant MitoTracker Red CMXRos (ThermoFisher, M7512) for 30 min at 37℃ in a dark environment and 5% CO2 in air. After washing three times with fresh M2 medium for 20 min each, oocytes were observed under laser scanning con focal microscope.
Mitochondrial membrane potential was evaluated using JC-1 Apoptosis Detection Kit ( KGA1904). oocytes were cultured in M16 medium with JC-1 operating fluid for 30 min at 37 C, followed by washing with buffer for 10 min. Samples were immediately imaged in a glassbottom dish under the laser scanning confocal microscope. JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (530 nm) to red (590 nm). Thus, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.
ROS levels were detected in oocytes using the DCFH-DA (2,7-Dichlorofluorescin Diacetate) probe, following the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, E004-1-1). Briefly, after the oocytes were incubated in DCFH-DA working solution at 37°C for 20 min and washed twice with (phosphate-buffered saline) PBS, the ROS fluorescence signals in oocytes were collected and observed under the laser scanning confocal microscope.
For Annexin-V staining, oocytes or granulosa cells were stained with the Annexin-V Staining Kit (Beyotime, C1062) according to the manufacturer’s instruction. After washing twice in PBS, the viable oocytes were stained for 30 min in the dark with 90 mL of binding buffer containing10 mL of Annexin-V-FITC. Then oocytes were washed three times in DPBS containing 0.1% BSA and placed on glass slides and observed under the laser scanning confocal microscope.
Evaluation of total ATP content
Total ATP content in a pool of 10–30 oocytes was determined using the Bioluminescent Somatic Cell Assay Kit, following the procedure described by Combelles and Albertini (2003) and the manufacturer’s instruction. A 5-point standard curve (0, 0.1, 0.5, 1.0, 10, and 50 pmol of ATP) was generated in each assay and the ATP content was calculated by using the formula derived from the linear regression of the standard curve.
Construction of ovario-targeted exosomes
FSHβ81–95 (DSPE-PEG2000-QCHCGKCDSDSTDCT) was synthesized by Qiang Yao Biotechnology Co., LTD. FSHβ was modified to the exosome surface. 10µL PBS containing Exosome (1012 particles /mL) and 90µL DSPE-PEGMal-FSHβ solution (10µM) were added to 100µL PBS and incubated overnight at 4℃. In order to remove the free DSPE-PEG-Mal-FSHβ, the mixture was centrifuged for 70min at 4°C with a centrifugal force of 120,000g in a ultra-high speed centrifuge, and the precipitation was suspended, thus the ovarian targeted modified exosomes were obtained.
Loading of exosome LIPE-AS1
10µL PBS (1012 particles /mL) containing exosomes and 100µg RNA fragments were mixed in 400µL electroporation buffer (1.15mM potassium phosphate pH 7.2, 25mM potassium chloride, 21% Opti-MEM reduced serum medium). The mixture was placed in a 4mm shock cup, cooled on ice for 6min, and then electroperforated at 0.35s, 20 pulses, and 0.7kV by an electroperforator. After completion, the exosomes were placed at 37°C for 30 min to restore the complete membrane structure of the exosomes. In order to remove the free RNA fragments, the resulting products were subjected to 1.2×105g ultra-high speed centrifugation at 4°C for 90min.
Statistical analysis
Statistical analysis was performed with GraphPad Prism software (GraphPad Software, San Diego, CA). The results are given as the means and standard deviations (SDs). Each experiment included at least three independent samples andwas repeated at least three times. Student’s two-sided t-test was used to calculate the difference between two groups, while difference among multiple groups was calculated by One-way analysis of variance (ANOVA). P values of < 0.05 were considered to indicate statistical significance. The sample sizes (“n”), the statistical tests and the P values were described in each figure legend.