Transcriptomic responses in zebrafish ovary to PNX and receptor agonists

Zebrafish ovary in vitro assays were completed using methods modified from Rajeswari et al., 2020. Briefly, female D. rerio (n = 14) were euthanized in 300 mg/L buffered MS-222 (Syncaine®, tricaine methanesulfonate, Syndel, Ferndale, WA, USA), weighed, and measured (total length (mm), TL). Zebrafish ovaries from each fish were partitioned into 5 sections of approximately equal size. Each of four of these sections were immersed into an assigned well within a 24-well plate containing 990 µl of culture media (90% Leibovitz's L-15 medium [Sigma Cat# L1518], 1% Penicillin-Streptomycin [Gibco™ Cat# 15070-063], 0.5% bovine serum albumin [Sigma Cat# 81053N]), with either a DMSO control or one of 3 potential agonist treatments, 8535 (5 µM), PT-91 (5 µM), or PNX-20 (100 nM). Agonists were dissolved in DMSO and added to their respective wells in 10µl aliquots to achieve desired final concentrations in a final volume of 1ml in each well. The fifth section of ovary was photographed on a Sedgewick rafter cell with a Jenoptik ProgRes® C5 microscope camera to characterize egg stages for each fish. Four plates containing n=3-4 replicates per treatment were incubated for 6 hours at 28 ?, each plate completed on different days to replicate the assay temporally. After incubation, ovarian tissues were snap frozen in RNAlater™ (Invitrogen Cat# AM7020) for transcriptome analysis and culture media were stored in glass tubes at -80°C for further steroid quantification analysis via liquid chromatography tandem mass spectrometry (LC-MS/MS). Overall design: 16 RNA-seq libraries, n=4 fish/treatment (control, A8535, PNX, PT91)