In the spawning seasons (late booleaf wrasse had been stuck because of the hook and you may line for the seaside oceans close to the Fisheries Look Laboratory, Kyushu School and transferred to the newest lab. Fish was kept in five-hundred-litre fiberglass tanks that have filtered seawater, lower than pure go out-size and you can liquids heat, and given krill and you can real time hermit crab once a day. After guaranteeing each and every day spawning, 4–six females fish (lbs – g, full length 11step 3–159 mm) was indeed sampled at , , , and hr. Seafood were anesthetized with dos-phenoxyethanol (300 ppm), and you can bloodstream samples was basically gathered throughout the caudal ship having fun with syringes suitable which have twenty-five-grams to own 20 minute. The fresh split up serum is actually kept at the ?30°C up until assayed to own steroid level. Immediately after bloodstream testing, fish had been murdered of the decapitation, in addition to ovaries were dissected away. To own ovarian histology, quick ovarian fragments had been fixed inside the Bouin’s solution, dehydrated, and you can stuck when you look at the Technovit resin (Kulzer, Wehrheim). New developmental grade from oocytes have been in the past stated (Matsuyama et al., 1998b).
The new developmental amount of one’s largest oocytes from the fish built-up within , , and you may hr was in fact tertiary yolk (TY), very early migratory nucleus (EMN), and later migratory nucleus (LMN) amounts, correspondingly. The greatest follicles on the fish sampled from the hr, in which germinal vesicle description (GVBD) had currently happened additionally the cytoplasm are transparent on account of yolk proteolysis and you may hydration, was indeed also known as adult (M) phase.
For white microscopy, 4-?m-thick sections was basically reduce and discolored that have step 1% 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 bilgisayara older women dating indir 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).