Inside vitro hair follicle incubation having radiolabeled steroid precursors

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

When you look at the spawning 12 months (later booleaf wrasse was in fact trapped from the hook and you can range in the seaside oceans near the Fisheries Research Lab, Kyushu University and transferred to the fresh new laboratory. Seafood had been kept in 500-litre fiberglass tanks having blocked seawater, lower than pure date-duration and you will drinking water temperatures, and you may given krill and you can real time hermit crab daily. After verifying everyday spawning, cuatro–six people seafood (lbs – grams, total length 113–159 mm) had been sampled from the , , , and you may hour. Seafood was in fact anesthetized that have dos-phenoxyethanol (3 hundred ppm), and you will bloodstream trials were compiled on the caudal boat having fun with syringes fitting that have twenty five-g having 20 minute. The brand new broke up gel try held within ?30°C up until assayed to have steroid level. Just after blood sampling, fish had been murdered by decapitation, plus the ovaries was basically dissected out. To have ovarian histology, quick ovarian fragments was repaired in Bouin’s service, dehydrated, and embedded in the Technovit resin (Kulzer, Wehrheim). Brand new developmental values regarding oocytes was in earlier times said (Matsuyama et al., 1998b).

This new developmental degrees of one’s largest oocytes on fish collected during the , , and hr had been tertiary yolk (TY), very early migratory nucleus (EMN), and you can late migratory nucleus (LMN) level, correspondingly. The greatest hair follicles about seafood sampled during the hr, where germinal vesicle description (GVBD) had currently taken place additionally the cytoplasm try clear on account of yolk proteolysis and you may hydration, was indeed referred to as mature (M) phase.

To possess white microscopy, 4-?m-heavy areas was basically clipped and you may tarnished having step one% 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 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).

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