Gnrha

Blood, pituitary, and gonad samples were collected from both sexes of SYC during the different induced spawning events. Fully ripen SYC, of both sexes, were induced by administering GnRHa (Sigma, St. Louis, MO, USA), at a dose of 100 ng/100 g-bw in females and 10 ng/100 g-bw in males, for final gonadal maturation. After the administration of GnRHa, male and female fish were kept in a circular spawning tank. A continuous flow of seawater and oxygen was maintained. Spawning occurred approximately 24 h after induction. Tissue sampling was performed at different stages, such as before spawning, or just before induction of GnRHa (BSW), after 24 h of GnRHa administration, or during the release of gametes or spawning (DSW), and post-spawning (PSW). The PSW samples were collected after 7 days of spawning. Samples were collected from 10 fish for each sex and at each sampling point. After anesthetization, blood, pituitaries, and gonads were collected and stored.

Brain samples were collected during different induced spawning events of small yellow croaker of both sexes. Small yellow croaker at gonadal ripen stage were injected with GnRHa (Sigma, St. Louis, MO, USA) at a dose of 10 μg/100 g-bw (gram-body weight) for final gonadal maturation and reared in a circular tank with running seawater and continuous oxygen supply. Release of gametes was started after 24 h of injection. Brain tissues of both male and female fish were collected at three different time-points: before injection of GnRHa termed as before spawning (BSW), after 24 h of injection or during release of gametes or spawning (DSW) and at 7 days after releasing gametes or post-spawning (PSW).

GnRH analog (GnRH-a), Tetradecanoylphorbol acetate (TPA), Okadaic Acid, Polyethylenimine (PEI), 4′6-diamino-2-phenylindole (DAPI) and PLA kit were obtained from Sigma (Rehovot, Israel). GF109203x was obtained from Calbiochem (Darmstadt, Germany). Protein A/G beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dharmafect was obtained from Thermo Scientific (Lafayette, CO, USA). 8-iso PGF2 α was purchased from Cayman Chemical (Ann Arbor, MI, USA). Monoclonal anti-PP2A Ab was obtained from BD Transduction Laboratories (New Jersey, USA). Anti-IGBP1 and monoclonal anti-PI3K Abs were obtained from Abcam (Cambridge, UK). Anti-GFP and anti-HA Abs were obtained from Roche Diagnostics (Mannheim, Germany). Monoclonal anti-AKT, Tubulin, GAPDH, and PKC α were obtained from Santa Cruz Biotechnology (CA, USA). Abs to phosphorylated JNK (pJNK), general JNK1/2 (gJNK), general AKT (gAKT) and histone H1 were from Sigma (Rehovot, Israel). Anti-phospho AKT (pS473AKT) was obtained from Cell Signaling Technology (Boston, MA, USA). Anti-PI3K was purchased from Upstate (Lake Placid, NY New York), or Millipore (Billerica, MA). The anti-phosphorylated PI3K (P85-S608) Abs was prepared by the Ab unit of the Weizmann Institute of Science (Rehovot Israel as previously described [50 (link), 51 (link)]). Secondary Ab conjugates were from Jackson Immunoresearch (West Grove, PA, USA).