Animals and clinical diagnosis of ovarian cysts
All cows were from local dairy farms in Beijing. First, the ovarian conditions of nonpregnant lactating cows were monitored daily by ultrasonography using a Honda HS1600V real-time B-mode scanner equipped with a 7.5 MHz linear-array trans-rectal transducer (Honda, Toyohashi, Japan). Based on the ultrasound image, in the absence of active luteal tissue, a follicle structure diameter >20 mm for more than 15 days with a thin wall (≤3 mm) and uniformly anechogenic follicular fluid was defined as a follicular cyst, while a thicker wall (>3 mm) with a visible echogenic rim, spots, and web-like structures signified a corpus luteum cyst (25). Holstein cows with normal preovulatory follicles on day 18 after synchronization of estrus and without reproductive disorders were assigned to the control group.
Tissue sampling, follicular fluid collection, and processing
Following B-ultrasound diagnosis, cyst and preovulatory follicles were obtained from cows during slaughter at a nearby abattoir, and rinsed in ice-cold saline (0.9% NaCl). The follicular fluid (one sample collected from 1 cow) of the upper follicles was extracted, centrifuged at 2000 rpm for 10 min, the supernatant was collected in sterilized Eppendorf tubes, the follicular wall was exfoliated and immediately preserved in liquid nitrogen, and samples were stored at -80°C until further analysis.
Measurement of hormone concentrations in follicular fluid
ELISA was conducted to measure the concentrations of oestradiol, P4, insulin and IGF-1 levels in follicular fluids. All ELISA kits were purchased from the Jiancheng Bioengineering Institute (Nanjing, China), and assays were performed according to the manufacturer’s instructions. Briefly, 50 μL of standards or samples were added to the appropriate well of the microtiter plate pre-coated with antibody, gently mixed, and incubated for 60 min at 37°C. After washing, biotinylated anti-IgG and streptavidin-horseradish peroxidase (HRP) were added along with chromogen solutions A and B. Finally, the optical density (OD) at 450 nm was recorded using a Multiskan MK3 automatic microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Hormone concentrations were calculated according to standard curves, and each experiment was repeated independently at least three times.
RNA extraction, library preparation, and sequencing
Total RNA was extracted from follicular walls using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and RNA was purified using an miRNeasy kit (Qiagen, Hilden, Germany). Sequencing and bioinformatics analysis were conducted by Beijing Genomics Institute (BGI; Beijing, China). The quality of RNA was assessed using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Palo Alto, CA, USA) and samples with a RNA integrity number (RIN) >8 were used for RNA library construction. Briefly, polyA+ mRNA was purified using oligo-dT-attached magnetic beads. Selected mRNAs were fragmented and reverse-transcribed to double-stranded cDNA (dscDNA) using N6 random primers. Ends of dscDNAs were repaired with phosphate at the 5’ end and A at the 3’ end to ligate with adaptors with T at the 3’ end of dscDNAs, which were subjected to amplification (26). RNA-seq libraries were sequenced on a BGISEQ-500 instrument (BGI; www.genomics.org.cn). Detailed procedures have been published previously (27). Raw reads have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA602176。
Bioinformatics analysis
Raw RNA-seq data were filtered into clean reads, followed by mapping against the Bos taurus reference genome (mm10) using HISAT (28). Gene expression levels were quantified using the RSEM software package (29). DEGs between control and cyst groups were identified using fold change ≥2 and false discovery rate (FDR) ≤0.001 as criteria. Gene Ontology (GO) annotation was used to map all DEGs to GO terms in the database (http://www.geneontology.org/), and GO terms with Q-values (corrected p-value) ≤0.05 defined DEGs as significantly enriched. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to perform pathway enrichment analysis of DEGs, and pathway terms with Q-values ≤0.05 were defined as significantly enriched (30).
Statistical analysis
All data are presented as mean percentages ± standard error of the mean (SEM) from a minimum of three independent replicate experiments. Different groups were analyzed by SPSS version 12.0 (SPSS, Chicago, IL, USA) and significant differences between means were determined using the least significant difference (LSD) test for comparison of multiple means. Statistical significance was defined at p <0.05.