Seafood and you may testing

Inside the spawning 12 months (later booleaf wrasse were trapped from the hook and range inside the seaside seas close to the Fisheries Lookup Research, Kyushu School and you will relocated to the new research. Fish was indeed kept in five-hundred-litre fiberglass tanks which have blocked seawater, less than natural go out-size and water temperature, and you may fed krill and you may real time hermit crab daily. Immediately following guaranteeing everyday spawning, 4–six people seafood (weight – g, overall duration 113–159 mm) had been tested at , , , and hours. Fish have been anesthetized with 2-phenoxyethanol (3 hundred ppm), and you will blood samples was in fact gathered about caudal vessel using syringes fitting that have twenty-five-grams getting 20 min. The latest split serum was kept in the ?30°C until assayed to own steroid level. Shortly after bloodstream sampling, fish was slain of the decapitation, and the ovaries were dissected aside. To have ovarian histology, short ovarian fragments was indeed fixed into the Bouin’s provider, dehydrated, and you may inserted inside Technovit resin (Kulzer, Wehrheim). The fresh developmental levels away from oocytes was in fact before said (Matsuyama et al., 1998b).

Brand new developmental grade of one’s prominent oocytes in the seafood compiled during the , , and hour was basically tertiary yolk (TY), early migratory nucleus (EMN), and late migratory nucleus (LMN) degrees, correspondingly. The greatest hair follicles from the seafood sampled at hr, in which germinal vesicle dysfunction (GVBD) had already took place additionally the cytoplasm try transparent on account of yolk proteolysis and you can moisture, was indeed known as adult (M) phase.

Having white microscopy, 4-?m-heavy sections was in fact slashed and you can tarnished with step 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 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 vanilla umbrella giriÅŸ 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).