Inside the spawning year (late booleaf wrasse was basically trapped because of the link and range when you look at the coastal oceans around the Fisheries Search Lab, Kyushu School and you will transferred to new laboratory. Seafood were kept in five hundred-litre fiberglass tanks which have filtered seawater, lower than pure go out-length and liquids temperatures, and you will provided krill and live hermit crab once a day. Shortly after verifying each day spawning, 4–6 female fish (lbs – grams, total duration 11step 3–159 mm) was basically sampled within , , , and you can hour. Fish was basically anesthetized which have dos-phenoxyethanol (3 hundred ppm), and you can blood examples was built-up regarding the caudal motorboat having fun with syringes installing with 25-g getting 20 minute. The separated solution are kept during the ?30°C up to assayed to have steroid top. Immediately after bloodstream testing, fish was killed by decapitation, and the ovaries was in fact dissected away. Getting ovarian histology, quick ovarian fragments were repaired when you look at the Bouin’s solution, dehydrated, and you will embedded into the Technovit resin (Kulzer, Wehrheim). Brand new developmental amounts off oocytes was prior to now said (Matsuyama et al., 1998b).
The new developmental amount of your premier oocytes in the seafood compiled in the , , and you may hour was tertiary yolk (TY), early migratory nucleus (EMN), and you will later migratory nucleus (LMN) stages, correspondingly. The most significant hair follicles from the seafood sampled at the time, in which germinal vesicle breakdown (GVBD) had already took place therefore the cytoplasm was transparent on account of yolk proteolysis and you can moisture, was basically referred to as adult (M) phase.
Having light microscopy, 4-?m-dense sections was slash and you can discolored with 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).