Inside the spawning 12 months (later booleaf wrasse was indeed trapped by hook up and you can range in coastal oceans close to the Fisheries Lookup Lab, Kyushu College and moved to the fresh lab. Fish have been stored in five hundred-litre fiberglass tanks that have filtered seawater, less than natural big date-size and you may h2o heat, and you can given krill and you will real time hermit crab once a day. Just after guaranteeing daily spawning, cuatro–6 people fish (fat – g, full duration 113–159 mm) were sampled on , , , and hour. Seafood was indeed anesthetized which have 2-phenoxyethanol (3 hundred ppm), and you may blood samples was indeed built-up about caudal watercraft playing with syringes suitable having twenty five-grams to own 20 minute. Brand new split gel is actually held within ?30°C up until assayed to possess steroid level. Immediately after bloodstream sampling, fish had been killed by decapitation, plus the ovaries was basically dissected away. To own ovarian histology, small ovarian fragments was indeed repaired inside the Bouin’s service, dried, and you will stuck from inside the Technovit resin (Kulzer, Wehrheim). The latest developmental values away from oocytes was in fact in the past claimed (Matsuyama et al., 1998b).
The fresh new developmental amounts of the largest oocytes throughout the fish obtained on , , and you can hours was basically tertiary yolk (TY), very early migratory nucleus (EMN), and you can late migratory nucleus mobifriends nasÄ±l Ã§alÄ±ÅŸÄ±r? (LMN) grade, respectively. The greatest hair follicles about seafood sampled at the hr, where germinal vesicle malfunction (GVBD) had currently happened while the cytoplasm is actually clear on account of yolk proteolysis and you can moisture, was named adult (M) stage.
Getting light microscopy, 4-?m-heavy areas had been slashed and discolored with step one% toluidine blue soluton
Ovarian follicles collected at hr were used for in vitro incubation with radiolabeled steroid precursors. After decapitation, the ovaries were removed and placed in ice-cold Ringer’s solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 1.5 mM NaH2PO4, 2 mM NaHCO3, 20 mM Hepes, pH adjusted to 7.5 with 1 N NaOH). The largest follicles (n=250) were isolated and gathered with forceps and pipettes. After removing of excess solution, follicles were frozen in liquid nitrogen and stored at ?80°C until use. Our preliminary experiments revealed that there was little difference in the steroid metabolic patterns during the incubation with frozen and intact follicles.
250 follicles were placed in a 10-ml glass tube with 1 ml of sucrose buffer (250 mM sucrose, 20 mM Hepes, pH adjusted to 7.6 with 1 N NaOH). Ten pmol of [ 3 H]P5, [ 3 H]17-P, [ 14 C]DHEA, [ 14 C]AD, [ 14 C]T, or [ 3 H]E1 were dissolved in 150 ?l sucrose buffer. Coenzymes (NAD, NADH, NADP, and NADPH; 10 mM each) were dissolved in a solution that consisted of 100 ?l MgCl2 (20 mM) and 50 ?l citrate buffer (5 mM, pH 7.3). At the start of incubation, both radiolabeled precursor and coenzymes solutions were added to the incubation media. Incubations were performed at 20°C for 2 hr with constant shaking. At the end of incubation, steroids were extracted three times from the media with 4 ml dichloromethane. The extract was concentrated and applied to a thin layer chromatography (TLC) plate (60F254; Merck, Darmstadt, Germany) with non-radioactive standard steroids, i.e., E1, E2, AD, T, progesterone, 17-P, and 17,20?-dihydroxy-4-pregnen-3-one (17,20?-P), and then developed in benzene:acetone (4:1). Radioactive steroid metabolites were analyzed with a BAS 1500 bio-imaging analyzer (Fuji Film, Tokyo), and standard E1 and E2 were visualized by exposure to iodine vapor. Other standard steroids were detected by UV absorption at 254 nm. Radioactive steroids were scraped from the TLC plates and extracted three times with 3 ml diethyl ether. Some radioactive metabolites were further separated in different solvent systems. Radiolabeled steroid metabolites were identified by their chromatographic mobility in TLC and by recrystallization as described by Axelrod et al. (1965).