Ethics statement and patients
This study was approved by and performed in accordance with the Ethics Committee of ZhuJiang Hospital of the Southern Medical University. All patients gave written informed consent for whole-exome sequencing.
A total of 20 unrelated Han Chinese women were recruited. All had at least 6 months of amenorrhea before the age of 40, high FSH plasma levels (>25 IU/mL) twice at least four weeks apart, and a normal 46 XX karyotype. Women having a background of pelvic surgery, anticancer treatment, ovarian infection, autoimmune disease, and/or positive family history were excluded from this study. The clinical characteristics of 20 POI patients are summarized in detail in Supplemental Table 1.
WES and bioinformatics analysis
Genomic DNA from patients was extracted from peripheral blood leukocytes using standard procedures. WES was performed on patients 1-10 using the SureSelect Human All Exon V6 /V6+UTR Kit (Agilent Technologies, Santa Clara, CA, USA) on the NextSeq 500 platform from Illumina (San Diego, CA USA). The raw image files were processed into variants with high reliability through several steps of base calling, quality control, alignment, and calibration. Basic sequencing information, including bases, length of reads, depth, and coverage is available in Supplemental Table 2. Single nucleotide variants and indels were then annotated using ANNOVAR. Variants fulfilling the following criteria were retained: (1) absent or rare variants (frequency < 0.01) in the dbSNP, 1000 Genomes Project, and Exome Aggregation Consortium databases; (2) protein structure-altering variants with pathogenicity predicted by SIFT, PolyPhen2, Mutation Assessor, and Mutation Taster.
Validation of mutation by Sanger sequencing
The mutations identified by WES and selected after literature reference were verified by Sanger sequencing in all 20 patients. Primers were designed to amplify the target region flanking the mutation site and are listed in Supplemental Table 3. Purified PCR products were sequenced on an ABI 3730XL (Applied Biosystems, USA) using the BigDye 3.1 Terminator Sequencing Kit (Applied Biosystems, USA) following the manufacturer's protocol.
Induction of follicle development and isolation of ovaries in rats
To stimulate follicular growth, immature female Sprague-Dawley (SD) Rats (24-25 days) were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG, 40 IU/rat) (Ningbo Second Hormone Plant, Zhejiang, China). The experimental group (PMSG group, n=3) was treated as above. The control group (NS group, n=3) was intraperitoneally injected with an equal volume of normal saline (NS). Rats were sacrificed 48 h later, and the ovaries were removed immediately and cleaned with phosphate-buffered saline (PBS) for subsequent assays. One ovary of each rat (n=6) was used for RT-PCR, and one for western blotting.
Ovaries of mature female SD rats (12 weeks) were removed, formalin fixed, paraffin embedded, sectioned (4 μm), deparaffinized, and rehydrated. Endogenous peroxidase activity was blocked by incubation of the sections with 3% H2O2 for 15 min. Nonspecific binding was blocked with 5% bovine serum albumin (CWBIO, Jiangsu, China) for 30 min. After washing, sections were incubated overnight at 4℃ with an antibody against GPSM1 (1:200, Proteintech, Wuhan, China), followed by incubation with a biotinylated secondary antibody (1:1000) for 1 h at room temperature. After washing, the antibody complexes were visualized with a DAB Kit (ZSGB-BIO, Shanghai, China) according to the manufacturer’s instructions. The sections were counterstained with hematoxylin, then dehydrated, and mounted.
Isolation and culture of primary rat GCs
PMSG was intraperitoneally injected into immature female SD rats aged 24-25 days at 40 IU/rat. The rats were sacrificed 48 h later and the ovaries were immediately removed. After washing with PBS, the ovaries were placed in DMEM/F12 medium. GCs were released from the ovarian follicles into the medium by a syringe needle under an anatomic microscope, and then purified by filtration with a 200-μm stainless steel mesh. The isolated GCs were centrifugated at 1000 × g for 5 min and then resuspended in medium. The GCs were seeded in 6-well plates (1 × 106 cells/well) and cultured in DMEM/F12 containing 1% Penicillin/ Streptomycin and 15% fetal bovine serum at 37 C with 5% CO2 for 48 h to allow cells to attach.
Synthetic small-interfering RNAs (siRNAs) were purchased from Sangon Biotech (Shanghai). The sequences of Gpsm1-siRNA were 5'-CCUGCGGCACCUUGUCAUUTT-3', 5'-GCCUAUGGCAACCUGGGUATT-3', and 5'-CCGAUUCGAUGAGGCAAUUTT-3', named siRNA-1, siRNA-2, and siRNA-3, respectively. The sequence of siRNA for the negative control was 5'-UUCUCCGAACGUGUCACGUTT-3', named NC-siRNA. The siRNAs were delivered into the cells with Lipofectamine 3000 (Life Technology, Invitrogen, USA) according to the protocols supplied. The RNA and protein samples were collected 48 h and 72 h after transfection, respectively.
RNA isolation and RT-PCR
Total RNA was isolated from ovarian tissues or cultured GCs using TRizol reagent (TaKaRa, Japan) according to the manufacturer’s instructions. The first-strand cDNA for total RNA was synthesized using PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Japan). The expression levels of mRNA were detected by TB Green Premix Ex Taq II (TaKaRa, Japan) on a Bio-Rad Real-Time PCR system (Bio-Rad Inc, USA). Gapdh was used as the internal mRNA control. Target gene expression was determined using the 2−ΔΔCt method. The primer sequences for amplification are listed in Supplemental Table 4.
Western blot analysis
Total proteins were extracted from ovarian tissues or cultured GCs using RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) containing 1% phosphatase inhibitor and 1% protease inhibitor. The protein samples (15 μg) were loaded onto the sodium dodecyl sulfate polyacrylamide gel for electrophoresis, and then transferred to polyvinylidene difluoride membranes (Merck Millipore, Germany). After blocking in 5% non-fat milk at room temperature for 2 h, primary antibodies against GAPDH (1:500, Proteintech, Wuhan, China), GPSM1 (1:1000, Proteintech, Wuhan, China), PKAc (1:1000, Cell Signal, Beverly, MA, USA), CREB (1:1000, Cell Signal, Beverly, MA, USA), p-CREB (1:1000, Abcam, Cambridge, UK), Bcl-2 (1:1000, AbSci, WA, USA), Bax (1:1000, Proteintech, Wuhan, China), Caspase-3 (1:1000, Cell Signal, Beverly, MA, USA), and cleaved caspase-3 (1:1000, Cell Signal, Beverly, MA, USA) were incubated at 4℃ overnight. The membranes were then incubated with secondary antibodies (1:500, Proteintech, Wuhan, China) for 1 h at room temperature. ECL detection reagent (Merck Millipore, Billerica, MA, USA) was used to visualize the bands. All experiments were repeated at least 3 separate times.
Cell Counting Kit-8 (CCK8) assay
Transfected GCs (2×103) were plated into a 96-well plate. At 24, 48,72, 96, and 120 h, cells were treated with 10 μL CCK-8 solution (Corning, Beijing, China) per well. The absorbance value (OD value) of each well was measured at 450 nm on a microplate reader after incubation at 37℃ for 2 h. Each group was established in 5 wells. The proliferation rate was calculated and the cell proliferation curve was drawn. All tests were repeated at least 3 times.
Proliferation rate (%) = (mean OD value at detection time point – OD value in blank group) / (mean 24 h OD value – 24 h OD value in blank group).
Flow cytometry-based annexin/ propidium iodide (PI) assay
GCs seeded in 6-well plates were transfected with siRNA and cultured for 48 h. Then 1 × 106 cells were collected, washed twice with ice-cold PBS, and resuspended in binding buffer containing Annexin V-FITC and PI. After incubating for 30 min in the dark, cells were analyzed using a BD FACSVerser flow cytometer system (BD Biosciences, USA) equipped with BD FACSuite software.
All data were analyzed using SPSS software (Version 22.0, SPSS Inc., Chicago, USA), and the results are presented as mean ± SEM using at least 3 independent experiments. An unpaired Student t-test was performed when comparing two groups and one-way ANOVA was performed when comparing more than two groups. A P value of 0.05 or less was considered statistically significant (*P < 0.05, **P < 0.01).