When you look at the spawning season (late booleaf wrasse was indeed caught by the connect and range during the seaside waters around the Fisheries Browse Laboratory, Kyushu College or university and you can moved to brand new research. Seafood had been stored in 500-litre fiberglass tanks that have filtered seawater, less than sheer date-size and you may liquids heat, and you may provided krill and real time hermit crab once a day. Just after confirming day-after-day spawning, cuatro–6 lady fish (fat – grams, total duration 11step three–159 mm) was indeed tested at , , , and you can hour. Seafood was anesthetized that have 2-phenoxyethanol (three hundred ppm), and you can blood trials was basically built-up throughout the caudal vessel having fun with syringes suitable with 25-g getting 20 minute. The newest separated solution try kept on ?30°C up to assayed getting steroid height. Shortly after bloodstream testing, fish was basically murdered of the decapitation, plus the ovaries was indeed dissected out. For ovarian histology, short ovarian fragments have been fixed into the Bouin’s provider, dried, and you can embedded inside the Technovit resin (Kulzer, Wehrheim). The new developmental amount out-of oocytes had been in earlier times reported (Matsuyama et al., 1998b).
The brand new developmental level of premier oocytes on fish collected at the , badoo, and you may hr have been tertiary yolk (TY), very early migratory nucleus (EMN), and you can late migratory nucleus (LMN) degrees, correspondingly. The greatest hair follicles regarding seafood sampled on time, in which germinal vesicle dysfunction (GVBD) had currently happened in addition to cytoplasm is transparent due to yolk proteolysis and you may hydration, was in fact known as adult (M) phase.
To possess white microscopy, 4-?m-heavy areas had been reduce and you can discolored which have step 1% toluidine bluish 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).