When you look at the spawning season (later booleaf wrasse have been trapped from the hook up and you can range in seaside seas around the Fisheries Look Research, Kyushu University and relocated to new laboratory. Fish was in fact stored in five-hundred-litre fiberglass tanks with filtered seawater, not as much as pure time-duration and you can liquid temperature, and provided krill and you will alive hermit crab once a day. After guaranteeing everyday spawning, 4–six women seafood (pounds – g, overall length 113–159 mm) was in fact tested from the , , , and hr. Fish were anesthetized having 2-phenoxyethanol (3 hundred ppm), and bloodstream products were obtained throughout the caudal vessel playing with syringes fitted which have 25-grams getting 20 min. The brand new split up gel is actually held on ?30°C up until assayed to possess steroid level. Shortly after bloodstream sampling, fish have been killed by decapitation, and also the ovaries have been dissected out. To possess ovarian histology, quick ovarian fragments was in fact repaired during the Bouin’s provider, dehydrated, and you will embedded into the Technovit resin (Kulzer, Wehrheim). Brand new developmental stages out of oocytes had been in the past advertised (Matsuyama et al., 1998b).
The brand new developmental amount of premier oocytes on fish amassed within , , and you will hour have been tertiary yolk (TY), very early migratory nucleus (EMN), and later migratory nucleus (LMN) levels, correspondingly. The largest follicles regarding fish sampled during the hours, where germinal vesicle dysfunction (GVBD) got currently taken place plus the cytoplasm are transparent on account of yolk proteolysis and hydration, have been described as adult (M) phase.
Having white microscopy, 4-?m-thicker areas had been reduce and you will discolored that have 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 https://datingranking.net/de/europaische-dating-sites/ 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).