Metabolomics analysis reveals a bile acid mediated ovarian failure induced by low temperature in zebrafish

Low temperature treatment and sample preparationThe selected zebrafish were uniform in weight and size. Starting at 27oC, the water temperature in three sets of the tanks were gradually cooled to 22°C, 20°C and 13°C respectively in a rate of 0.5oC/hr with one set of the tanks remaining at 27oC. The flow circulation systems were then activated at temperatures set at 27oC, 22oC, 20oC and 13oC, respectively. The fish were exposed to the discrete temperatures for 14 days under a light/dark cycle of 14h/10h. Four sets of fish were fed with freshly hatched Artemia twice daily, at 9:00 am and 6:00 pm. Six biological replicates for each temperature, with each replicate containing 5 fish were set up. Fish after distinct temperature treatments were sampled for ovary imaging, histological analysis, and LC-MS/MS measurement.Ultra-performance liquid chromatography-tandem mass spectrometry-based (LC-MS/MS) metabolomic analysis.Fresh ovarian tissues were rapidly frozen in liquid nitrogen and stored at –80℃. Subsequently, 100 mg of each tissue sample was homogenized individually in liquid nitrogen and the resulting homogenate was resuspended with pre-chilled 80% methanol containing 0.1% formic acid through vigorous vortexing. The samples were then incubated on ice for 5 minutes and subsequently centrifuged at 15 000 g, 4°C for 20 minutes. 400μL supernatant was further diluted to final concentration containing 53% methanol using LC-MS grade water. The resulting samples were transferred to fresh Eppendorf tubes and subjected to a second centrifugation step at 15 000 g, 4°C for 20 minutes. Finally, the supernatant was injected into the LC-MS/MS system for analysis. Take 10μL from each experimental sample and mix it evenly to create a quality control (QC) sample.Transcriptome AnalysisTotal RNA was extracted using Trizol reagent (Sigma). Strand-specific RNA-seq libraries were prepared using the VAHTS Stranded mRNA-seq Library Prep Kit for Illumina v2 (NR612-02, Vazyme Biotech Co., Ltd, Nanjing, China) and subjected to 150bp paired-end sequencing on the Illumina Novaseq 6000 platform. Trimmomatic software was employed to remove low-quality reads and sequencing adapters, followed by the alignment of clean reads to the zebrafish reference genome GRCz11 (v106) using STAR software. Read counts for each gene were calculated using featureCounts. Finally, Fragments per kilobase million (FPKM) values for each gene were obtained using DESeq2.