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In vitro hair follicle incubation having radiolabeled steroid precursors

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

Inside the spawning year (later booleaf wrasse were stuck by connect and range during the coastal oceans around the Fisheries Look Lab, Kyushu College or university and gone to live in the fresh lab. Seafood was stored in five hundred-litre fiberglass tanks with blocked seawater, significantly less than pure time-duration and you will drinking water temperatures, and you will given krill and you may real time hermit crab daily. Once confirming every single day spawning, 4–6 girls fish (weight – g, overall duration 11step three–159 mm) was indeed tested in the , , , and hr. Seafood was basically anesthetized having 2-phenoxyethanol (300 ppm), and bloodstream samples was collected on caudal vessel having fun with syringes fitting with 25-g to own 20 min. The fresh new separated gel was stored during the ?30°C up to assayed to possess steroid level. After blood testing, fish have been killed from the decapitation, together with ovaries had been dissected aside. To have ovarian histology, brief ovarian fragments was in fact fixed in the Bouin’s services, dehydrated, and stuck inside the Technovit resin (Kulzer, Wehrheim). New developmental amount of oocytes had been in the past stated (Matsuyama mais aussi al., 1998b).

New developmental values of one’s largest oocytes on the fish built-up at the , , and hr was tertiary yolk (TY), very early migratory nucleus (EMN), and you may later migratory nucleus (LMN) values, respectively. The greatest follicles throughout the seafood sampled at the hours, in which germinal vesicle dysfunction (GVBD) got already happened and also the cytoplasm was clear on account of yolk proteolysis and you can moisture, was in fact described as adult (M) stage.

To have light microscopy, 4-?m-thicker areas were cut and you will stained that 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 dissolved in 150 ?l sucrose buffer. Coenzymes (NAD, NADH, NADP, and NADPH; 10 mM each) were dissolved in a solution that consisted of 100 silversingles indirim kodu ?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).