Seafood and you can testing
Inside spawning year (late booleaf wrasse was indeed trapped by the connect and line into the seaside oceans close to the Fisheries Look Lab, Kyushu College or university and relocated to the brand new research. Seafood were stored in 500-litre fiberglass tanks with blocked seawater, lower than pure big date-duration and you will water heat, and you will provided krill and you can live hermit crab once a day. Once confirming daily spawning, 4–6 women fish (fat – g, total length 11step three–159 mm) was in fact sampled during the , , , and you will time. Fish was basically anesthetized having dos-phenoxyethanol (three hundred ppm), and bloodstream trials was obtained on caudal watercraft using syringes fitted having 25-grams for 20 minute. The latest split up solution is actually kept from the ?30°C until assayed to have steroid height. Once bloodstream sampling, seafood was murdered of the decapitation, and ovaries were dissected away. To possess ovarian histology, brief ovarian fragments was fixed when you look at the Bouin’s provider, dehydrated, and you may embedded in Technovit resin (Kulzer, Wehrheim). The brand new developmental amount from oocytes was indeed in past times advertised (Matsuyama mais aussi al., 1998b).
The brand new developmental stages of your own premier oocytes regarding seafood amassed from the , , and you will hr had been tertiary yolk (TY), early migratory nucleus (EMN), and you will later migratory nucleus (LMN) grade, respectively. The greatest hair follicles on fish tested on hours, in which germinal vesicle description (GVBD) got already taken place therefore the cytoplasm are transparent on account of yolk proteolysis and you may moisture, was in fact referred to as adult (M) phase.
Getting light microscopy, 4-?m-heavy areas have been cut and you may stained with 1% toluidine bluish 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 romance tale bio 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).