Inside the vitro hair follicle incubation having radiolabeled steroid precursors
Seafood and you can sampling

From inside the spawning seasons (late booleaf wrasse were trapped of the hook up and you will line into the seaside seas close to the Fisheries Browse Research, Kyushu College and you may transferred to the fresh new laboratory. Fish were kept in five-hundred-litre fiberglass tanks with blocked seawater, lower than pure date-length and you can water temperatures, and you may provided krill and you can alive hermit crab once a day. After verifying daily spawning, 4–six lady fish (body weight – grams, complete duration 11step three–159 mm) were tested in the , , , and you may hour. Seafood had been anesthetized which have 2-phenoxyethanol (three hundred ppm), and you may bloodstream samples had been compiled regarding caudal vessel using syringes suitable with twenty-five-g having 20 minute. The fresh split up serum is held at the ?30°C up until assayed to possess steroid top. Shortly after bloodstream sampling, seafood was basically murdered because of the decapitation, and also the ovaries were dissected out. Getting ovarian histology, brief ovarian fragments was basically fixed when you look at the Bouin’s solution, dehydrated, and you can stuck when you look at the Technovit resin (Kulzer, Wehrheim). Brand new developmental amounts regarding oocytes were prior to now said (Matsuyama ainsi que al., 1998b).

The newest developmental levels of the prominent oocytes in the fish built-up from the , , and you can hr was in fact tertiary yolk (TY), very early migratory nucleus (EMN), and you will later migratory nucleus (LMN) stages, respectively. The biggest hair follicles in the seafood tested during the hour, where germinal vesicle description (GVBD) had already took place and also the cytoplasm is transparent on account of yolk proteolysis and you will moisture, had been referred to as adult (M) stage.

Having white microscopy, 4-?m-dense sections was in fact reduce and you may discolored which have step 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 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).