In spawning year (later booleaf wrasse was basically stuck because of the hook and you will range within the seaside waters around the Fisheries Look Research, Kyushu College and you will relocated to the new lab. Fish was basically kept in five hundred-litre fiberglass tanks that have blocked seawater, lower than absolute go out-duration and water temperature, and you can provided krill and you may alive hermit crab daily. Just after confirming every day spawning, cuatro–six people seafood (pounds – grams, complete length 11step three–159 mm) was in fact sampled at the , , , and time. Seafood have been anesthetized which have 2-phenoxyethanol (300 ppm), and you can blood trials have been accumulated regarding caudal watercraft having fun with syringes fitted that have 25-g to own 20 minute. The brand new split up solution are held during the ?30°C up until assayed for steroid top. Immediately following bloodstream sampling, fish were murdered by the decapitation, as well as the ovaries were dissected out. Getting ovarian histology, short ovarian fragments was indeed repaired during the Bouin’s provider, dehydrated, and you may stuck during the Technovit resin (Kulzer, Wehrheim). The new developmental levels away from oocytes was in fact in the past reported (Matsuyama mais aussi al., 1998b).
The brand new developmental values of your own largest oocytes from the seafood obtained within , , and time have been tertiary yolk (TY), very early migratory nucleus (EMN), and you can later migratory nucleus (LMN) level, respectively. The biggest hair follicles about fish sampled from the hour, in which germinal vesicle description (GVBD) had currently took place together with cytoplasm is clear because of yolk proteolysis and you will hydration, was indeed known as adult (M) phase.
Having white microscopy, 4-?m-dense areas have been clipped and you will tarnished having 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 lumen dating ne demek 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).