Fish and you can sampling
During the spawning year (later booleaf wrasse was stuck by the hook and you may line from inside the seaside seas around the Fisheries Research Laboratory, Kyushu College or university and you may transferred to this new laboratory. Seafood was indeed kept in five-hundred-litre fiberglass tanks that have blocked seawater, not as much as natural go out-length and you may drinking water heat, and you can provided krill and you can live hermit crab daily. Once confirming daily spawning, cuatro–six lady seafood (body weight – g, complete length 113–159 mm) were sampled on , , , and you will hours. Fish were anesthetized that have dos-phenoxyethanol (three hundred ppm), and you will bloodstream samples were accumulated in the caudal watercraft using syringes fitted that have twenty five-grams to have 20 min. The fresh split up gel is actually held within ?30°C up to assayed having steroid height. Once blood sampling, seafood was murdered from the decapitation, and the ovaries had been dissected out. To possess ovarian histology, quick ovarian fragments was basically repaired for the Bouin’s services, dehydrated, and you may inserted within the Technovit resin (Kulzer, Wehrheim). The new developmental values away from oocytes had been previously claimed (Matsuyama et al., 1998b).
The newest developmental amount of one’s biggest oocytes on seafood obtained during the , , and you can time have been tertiary yolk (TY), very early migratory nucleus (EMN), and you will late migratory nucleus (LMN) levels, respectively. The largest hair follicles on seafood sampled at time, where germinal vesicle breakdown (GVBD) got currently happened in addition to cytoplasm are transparent on account of yolk proteolysis and you will hydration, had been referred to as adult (M) phase.
Having light microscopy, 4-?m-thick parts was slash and you will 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 ourtime online.
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).