The production of a viable vertebrate ovum (oogenesis) is a complex process during which maternal provisions support growth and development of the egg cell (oocyte) and surrounding somatic cell layers, together known as the ovarian follicle. Vertebrate sexual maturity is reached at the onset of puberty, demarcated by the shift of late primary growth ovarian follicles (i.e., early and late perinucleolar stages [EPN, LPN]) into early secondary growth (i.e., early cortical alveolus stage [ECA]). Gonadotropin (follicle stimulating hormone [FSH], luteinizing hormone [LH]) regulation of oogenesis after puberty has been well studied. Hormonal regulation of stages connected with the transition into puberty is less understood. Our research aimed to determine the regulatory role of gonadotropins at the primary and early secondary growth stages of oogenesis. Selected reaction monitoring (SRM) proteomics identified differentially expressed proteins in coho salmon (Oncorhynchus kisutch) ovarian fragments exposed to FSH and LH.

In vitro cultures using coho salmon (Oncorhynchus kisutch) ovarian explant tissue from six fish per stage (LPN, ECA; n = 12) were exposed to a total of five different treatments: no treatment, recombinant FSH (50 ng/ml or 500 ng/ml), or recombinant LH (50 ng/ml or 500 ng/ml) for 36 hours. Quantifiable peptides for proteins of interest for SRM were determined based on the discovery DIA mass spectrometry analysis found under ProteomeXchange ID PXD052361. Target proteins were imported into Skyline and proteins with  2 proteolytic peptides were selected for SRM analysis. SRM was carried out on a Thermo TSQ Altis mass spectrometer in two technical replicates for the ovarian samples exposed to GTHs. Sample preparation consisted of 12.5 µl coho salmon peptides, 1.25 µl of 50 fmol PRTC peptides, and 11.25 µl 2% acetonitrile for a total volume of 25 µl per sample. A new 5 cm trap (fused silica, 100 um internal diameter) and 30 cm column (fused silica, 75 um internal diameter) were packed with 3 µm C18 beads. Samples were injected by an EASY-nLC system at a volume of 4 µl to monitor target peptide transitions (n = 483). A chromatography gradient of 6 -75% solvent (80% acetonitrile + 0.1% formic acid) was applied for 30 minutes (total run time 52 minutes). Spectra acquisition was collected in centroid mode, with a cycle time of 1.2 seconds. Resulting raw data files were exported into Skyline for automated peak selection.

Coho salmon (Oncorhynchus kisutch) ovarian explant tissue.