Nexera x3

Four testicular fragments from a P7 HV-Tg rat were placed on a 1.5% agarose gel block without a PC device as described above. The first day of culture was designated as day 0. 30 µL of medium was collected from each well on days 3, 7, 14, 17, 21, 24 and 28. On days 7, 14, 21 and 28, the culture medium was fully changed after collecting the sample. The LC/MS/MS method package for cell culture profiling (version 2; Shimadzu, Kyoto, Japan) was used for the metabolomics analysis. Briefly, 40 µL of Milli-Q water, 10 µL of 0.5 mM isopropylmalic acid (333115, Sigma) as an internal standard, and 100 µL of acetonitrile (018-19853, Fujifilm, Tokyo) were added to 10 µL of standard or sample medium and vortexed. 50 μL of the supernatant of the sample after centrifugation at 15,000 rpm for 15 min at room temperature was collected and diluted with 450 μL of Milli-Q water, and 1 μL was introduced into an LC (Nexera X3; Shimadzu) with a Discovery HS F5-3 column (567503-U, Merck; 2.1 mm I.D. × 150 mm, 3 μm) connected to a tandem quadrupole mass spectrometer (LCMS-8060NX; Shimadzu). The chromatographic parameters were set according to the instruction manual (225-41626A; Shimadzu). LabSolutions Insight (Version 3.7 SP3; Shimadzu) was used to analyze the chromatographic data.

LC-QqTOFMS analysis was performed using a Nexera X3 high-performance liquid chromatograph (Shimadzu Co.) equipped with an LCMS-9030 mass spectrometer (Shimadzu Co.). Chromatographic separation was carried out by applying the same conditions of those for LC-QqQMS analysis. Ionization was performed by ESI in positive mode under the following conditions: nebulizer gas flow rate, 3 L/min; heating gas flow rate, 10 L/min; drying gas flow rate, 15 L/min; interface temperature, 300 °C; desolvation line temperature, 250 °C; heating bloc temperature, 400 °C; and interface voltage, 4 kV. The DDA mode was applied. The scan range of the trigger scan was m/z 200–800. Product ion spectra were acquired at CE of 35 ± 17 V over m/z 10–800. Precursor ions, fragment ions, and the corresponding formulas are listed in the supplementary information (Table S1).