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For the vitro hair follicle incubation with radiolabeled steroid precursors

For the vitro hair follicle incubation with radiolabeled steroid precursors
Seafood and sampling

Into the spawning year (later booleaf wrasse have been stuck because of the connect and line when you look at the seaside seas near the Fisheries Browse Research, Kyushu University and you will moved to the fresh new research. Seafood were kept in five hundred-litre fiberglass tanks which have blocked seawater, not as much as absolute big date-length and you may water temperature, and you may provided krill and you can alive hermit crab once a day. Shortly after confirming each and every day spawning, cuatro–6 people fish (lbs – grams, complete size 113–159 mm) were tested during the , , , and you can hr. Seafood have been anesthetized with dos-phenoxyethanol (300 ppm), and you may bloodstream trials had been gathered on caudal vessel having fun with syringes fitting having 25-g for 20 min. New broke up gel are held during the ?30°C until assayed to own steroid level. Immediately following bloodstream testing, seafood had been killed because of the decapitation, while the ovaries was basically dissected away. To possess ovarian histology, quick ovarian fragments have been fixed in the Bouin’s provider, dehydrated, and you may embedded from inside the Technovit resin (Kulzer, Wehrheim). The developmental stages from oocytes was in fact before stated (Matsuyama mais aussi al., 1998b).

The fresh new developmental values of prominent oocytes regarding fish amassed on , , and you can time was indeed tertiary yolk (TY), early migratory nucleus (EMN), and you may later migratory nucleus (LMN) degree, correspondingly. The most significant hair follicles throughout the fish sampled at the hour, where germinal vesicle description (GVBD) got currently taken place while the cytoplasm are clear due to yolk proteolysis and you may moisture, was indeed named adult (M) phase.

Having white microscopy, 4-?m-dense parts was in fact slash and you may stained with 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).

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