Early-life vitamin B12 orchestrates lipid peroxidation to ensure reproductive success via SBP-1/SREBP1 in Caenorhabditis elegans
Description
We employed GC–MS to determine whether any specific lipid species accounted for the occurrence of ∆nhr-114 infertility, which could be rescued by early-life B12 treatment.
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Method：For total lipid (TL) and TAG extraction, 5,000 young adults were harvested with PBS. Worm lysates prepared by sonication were mixed with 3 mL of chloroform:methanol (2:1) solution in a glass tube for 1 hour for extraction at room temperature. The organic phase was then mixed with 0.4 mL of 0.9% NaCl and spun to separate (2000 rpm for 5 min). One-quarter of the bottom organic phase (500 μL) was dried under nitrogen as the TL extract. The remaining 1.5 mL was used for further TAG extraction. TAG was separated through thin-layer chromatography TLC in hexane:ether:acetic acid (80:20:2, vol/vol/vol). Both the TL and TAG extracts were resuspended in 700 μL of 14% BF3/MeOH solution and heated at 75°C for 45 min to create fatty acid methyl esters (FAMEs). The fatty acids were determined with an Agilent 5977B GC/MSD system equipped with a 60 m × 0.25 mm× 0.2 μm HP-88 column (Agilent 112-8867), with helium as the carrier gas at 1 mL/min. Three to four biological replicates were used for gas chromatography (GC) analysis. Methylated C13:0 was used as an internal standard for quantitative analysis.