Participants and Granulosa cell collect
All PCOS women and control women were recruited from Reproductive Medical Center of Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of science and technology. The inclusive criteria of PCOS women are as follow: 1, diagnosed with PCOS; 2, the ages were not more than 34 years old; 3, Except IVF treatment, no other treatments were done in a month. The exclusive criteria of PCOS women; 1, diagnosed with hyperprolactinemia; 2, abnormal androgen secretion due to adrenal or ovarian tumors; 3, patients with uncorrected thyroid disease; 4, suspected Cushing’s syndrome; 5, using estrogen or oral contraceptives and other hormone drugs in recent one month; 6, using other drugs that affect reproductive function or metabolism in the past 2 months (such as anti-obesity drugs, anti-diabetes drugs and traditional Chinese medicine, etc.). Control women receiving IVF treatment were recruited owning to fallopian tube jam、intrauterine adhesions and male factors. The ages of control women were not more than 34 years old. Granulosa cell was collected by Percoll density-gradient centrifugation method. The follicle aspirate was collected and centrifuged at 200 g for 10 min. The supernatant was discarded and the sediment was collected and resuspended with PBS. The resuspended solution was pipetted into the 50 % Percoll gradient (lot number: P4937, sigma) of equal volume and centrifuged at 400 g for 20 min. Intermediate cell layer was collected and resuspended with PBS, which was centrifuged at 200 g for 10 min. Finally, the sediment was collected and deposited at -80℃ refrigerator.
Total RNA extraction
Total RNA was extracted by Trizol method. 1ml Trizol reagent (Invitrogen, cat. NO 15,596,026) was pipetted into each tube of sediment (granulosa cell) at room temperature for 5 min. After centrifuging at 12,000 g for 15 min, the supernatant was collected into another tube, mixed with 200ul chloroform. After remaining for 15 min at room temperature and centrifuging at 12,000 g 4℃ for 15 min, the water layer of supernatant was removed into a tube mixed with 0.5ml isopropanol, keeping for 10 min at room temperature. After centrifuging at 12,000 g 4℃ for 10 min, the supernatant was discarded and sediment of RNA was collected resuspended with 1 ml 75 % ethanol. After centrifuging at 8000 g 4℃ for 5 min, the supernatant was discarded and sediment of RNA was obtained. The RNA was dissolved with TE buffer and concentration of RNA was measured by Nanodrop™ OneCspectrophotometer (Thermo Fisher Scientific Inc). 1 % agarose gel electrophoresis was administrated to observe the integrity of the strip.
After the total RNA samples were up to standard, 5ug of total RNA was taken for subsequent experiments. KCTM Stranded mRNA Library Prep Kit (Catalog NO. DR08402, Wuhan Seqhealth Co., Ltd. China) was applied in RNA library preparation according to the manufacturer’s instruction. The mRNA was enriched by magnetic beads with oligo (dT). Then, fragment buffer was added to break the resulting mRNA into short fragments. A six base random primer was used to synthesize a single strand of cDNA using the mRNA fragment as a template, and then two-strand cDNA was synthesized by adding buffer, dNTPs and DNA polymerase I. After elution and purification, the terminus of double stranded cDNA was repaired with base and added with sequencing adaptor. The 5 ‘end of cDNA was connected to UID connector. The fragment was caught by magnetic beads and PCR amplification was performed by T100™ Thermal Cycler (BIO-RAD, USA). Agarose electrophoresis was used to detect the quality of the cDNA library. Qubit 3.0 with Qubit™ RNA Broad Range Assay kit (Life Technologies, Q10210) is used to quantify the cDNA library and determine whether the cDNA library concentration is suitable for the computer. After the cDNA library passed the quality inspection, the different cDNA library was sequenced on Illumina sequencer (Illumina NovaSeq 6000) according to the requirements of effective concentration and target offline data volume.
Data analysis of UID-mRNA-seq
Quality control of raw data included quality scores across all bases and sequence content across all bases. Raw data was filtered via Trimmomatic (version 0.36) and reads of low quality were discarded. Clean data was mapped into the reference genome of human via STAR software (version 2.5.3a). Reads mapped into the exon regions of gene were counted by feature Counts (Subread-1.5.1).
RPKM (Reads per Kilobase per Million Reads) was calculated and normalized to estimate gene expression. Differentially expressed genes were identified using the edgeR package (version 3.12.1) by R4.0.2. Gene expression differences were judged by p-value < 0.05 and fold-change > 2. KEGG enrichment analysis and Go enrichment analysis for differentially expressed genes were administrated by KOBAS software (version 2.1.1).
Quantitative Real-time PCR
Total RNA was extracted by Trizol method. Reverse transcription PCR was implemented by strand cDNA Synthesis kit (11141ES60, Yeasen, China) according to instruction. Quantitative Real-time PCR was implemented by qPCR kit (11201ES08, Yeasen, China) according to instruction. FDX1, forward primer: CTTTGGTGC ATGTGAGGGAA, reverse primer: GCATCAGCCACTGTTTCAGG. β-actin, forward primer: GTCCACCGCAAATGCTTCTA, reverse primer: TGCTGTCACCT TCACCGTTC. The 2−ΔΔCT method was used for data analysis.
Two PCOS models were implemented. Letrozole and Testosterone Propionate were applied into modeling PCOS. All rats included Wistar weighting 250~300g, purchased from the Hubei Provincial Center for Disease Control and Prevention, Wuhan, China and housed in SPF room of experimental animal center, Tongji Medical College, Huazhong University of science and technology. This animal experiment and all operations were approved by the Institutional Animal Care and Use Committee at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Wistar rats modeling with Letrozole were perfused with Letrozole 1 mg/kg via stomach, continuing for 21days. Control Wistar rats and model Wistar rats were sacrificed at the end of perfusing. Wistar rats modeling with Testosterone Propionate were injected with 10 mg/kg Testosterone Propionate, continuing for 6 weeks. Control rats and model rats injected with Testosterone Propionate were sacrificed at the end of injection. Ovary was picked up and fixed by paraformaldehyde to Paraffin embedding. All rats(n = 6 for each group) were euthanized by intraperitoneal injection with pentobarbital sodium.
After paraffin section of ovary dewaxing with xylene and antigen repair with Sodium citrate repair solution, the sections were put into 3 % hydrogen peroxide solution and incubated for 25 min at room temperature. After sections washing for 3 times (each time for 5 min), sections were covered with 10 % goat serum for 30 min. Then, goat serum was discarded and anti-FDX1 antibody (ab108257, Abcam, UK) was added to incubate for the whole night at 4℃. After washing for 3 times (each time for 5 min), sections were incubated with secondary antibody (ab6721, Abcam, UK) for 1 h at room temperature. DAB was added into the section to render color for 1 min. After DAB stain stopping, cell nucleus was stained with Hematoxylin for 1 min. After Hematoxylin stain stopping, sections were added with 1 % alcohol hydrochloride for several seconds and added with Ammonia to turns blue. Finally, the sections were dehydrated with Gradient concentration alcohol and mounted. The length of granulosa cell and quantitative analysis were measured by the software of Image Pro Plus(version 7.0). The unit of length is expressed as pixels.
Student’s t test analysis was applied if data was meeting with normal distribution and homogeneity of variances. Otherwise, Mann-Whitney U was applied. P value < 0.05 was considered significant. IBM SPSS 23 was used to analyze data and GraphPad prism 7 was applied to plot.