In the spawning year (later booleaf wrasse have been caught by link and you can range from inside the seaside oceans nearby the Fisheries Lookup Research, Kyushu School and relocated to new lab. Fish had been kept in five-hundred-litre fiberglass tanks having blocked seawater, not as much as absolute go out-length and you will water temperature, and you may fed krill and you may live hermit crab once a day. After confirming day-after-day spawning, cuatro–six girls fish (lbs – grams, total length 113–159 mm) was basically tested within , , , and hours. Fish was basically anesthetized that have 2-phenoxyethanol (300 ppm), and you can bloodstream products was in fact gathered throughout the caudal motorboat using syringes installing which have twenty five-grams to own 20 minute. The newest split gel is kept within ?30°C up to assayed to possess steroid peak. Once blood testing, fish was killed of the decapitation, together with ovaries were dissected out. To own ovarian histology, short ovarian fragments were fixed when you look at the Bouin’s service, dehydrated, and you will stuck when you look at the Technovit resin (Kulzer, Wehrheim). The developmental amounts out of oocytes were before stated (Matsuyama et al., 1998b).
New developmental amounts of your own flingster bio örnekleri premier oocytes on the seafood collected on , , and you will time was basically tertiary yolk (TY), early migratory nucleus (EMN), and late migratory nucleus (LMN) stages, respectively. The biggest hair follicles regarding the fish tested from the time, in which germinal vesicle malfunction (GVBD) got already took place together with cytoplasm is actually transparent because of yolk proteolysis and you may hydration, were called adult (M) phase.
To own white microscopy, 4-?m-thick parts have been slashed and stained that have 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).