Inside the spawning season (late booleaf wrasse was basically caught from the connect and you will line inside the seaside seas around the Fisheries Research Laboratory, Kyushu University and you will transferred to this new lab. Seafood were kept in five hundred-litre fiberglass tanks that have filtered seawater, not as much as pure time-length and you can liquids heat, and you may provided krill and real time hermit crab once a day. Immediately after confirming each day spawning, cuatro–6 lady seafood (pounds – g, total duration 11step 3–159 mm) was tested at the , , , and you will time. Fish was in fact anesthetized which have 2-phenoxyethanol (three hundred ppm), and blood products was indeed accumulated on the caudal motorboat playing with syringes suitable with twenty five-g to possess 20 min. The latest split up solution is kept from the ?30°C up until assayed to own steroid top. Once bloodstream sampling, seafood was basically slain by decapitation, and the ovaries have been dissected out. To have ovarian histology, brief ovarian fragments have been fixed for the Bouin's provider, dehydrated, and you can stuck inside the Technovit resin (Kulzer, Wehrheim). Brand new developmental amount out of oocytes were prior to now advertised (Matsuyama mais aussi al., 1998b).

The fresh developmental level of one's biggest oocytes about fish obtained at the , , and time had been tertiary yolk (TY), very early migratory nucleus (EMN), and you will late migratory nucleus (LMN) amounts, correspondingly. The most significant follicles about fish tested at the hr, where germinal vesicle breakdown (GVBD) had already took place therefore the cytoplasm was transparent on account of yolk proteolysis and you can hydration, was described as mature (M) phase.

For white microscopy, 4-?m-thicker areas was basically slash and stained which have 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 http://www.datingranking.net/tr/three-day-rule-inceleme/ 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).