When you look at the spawning season (late booleaf wrasse was in fact caught by the link and you may line inside coastal seas close to the Fisheries Browse Research, Kyushu College and relocated to the latest lab. Fish was basically stored in five hundred-litre fiberglass tanks having filtered seawater, around natural date-duration and liquid temperatures, and you will provided krill and you can alive hermit crab once a day. Shortly after confirming daily spawning, cuatro–six ladies seafood (lbs – g, complete size 11step three–159 mm) have been tested during the , , , and you can hours. Fish have been anesthetized that have 2-phenoxyethanol (300 ppm), and you will blood products was basically gathered throughout the caudal watercraft using syringes fitting with 25-grams to have 20 min. The new separated gel was kept at the ?30°C until assayed having steroid peak. Once bloodstream sampling, fish have been slain from the decapitation, additionally the ovaries was dissected away. For ovarian histology, quick ovarian fragments had been fixed when you look at the Bouin’s solution, dehydrated, and inserted in Technovit resin (Kulzer, Wehrheim). The newest developmental level off oocytes have been in the past claimed (Matsuyama mais aussi al., 1998b).
The newest developmental amounts of premier oocytes throughout the seafood amassed on , , and hour was in fact tertiary yolk (TY), very early migratory nucleus (EMN), and later migratory nucleus (LMN) amount, respectively. The greatest hair follicles throughout the fish sampled at the hour, where germinal vesicle breakdown (GVBD) had already happened additionally the cytoplasm is actually clear on account of yolk proteolysis and you will hydration, were described as mature (M) phase.
To possess white microscopy, 4-?m-thick parts were slash and you can discolored 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.
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 https://datingranking.net/pl/biggercity-recenzja/ 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).